As a major cancer type in females, cervical cancer has been explored in depth by researchers. HeLa is a cervical cancer cell line. Isorhamnetin is an O-methylated flavonol that is primarily extracted from sea buckthorn. In the present study, the anti-proliferative effect of isorhamnetin on HeLa cells was evaluated using a Trypan blue dye exclusion assay. Isorhamnetin inhibited the cell proliferation in a time- and dose-dependent manner. Flow cytometric analysis of the cell cycle distribution revealed that isorhamnetin inhibited the cell cycle progression of HeLa by causing G2/M phase arrest and decreasing the proportion of cells in G1 phase. In addition, western blot analysis was performed to evaluate the presence of certain cell cycle-associated proteins. It was demonstrated that isorhamnetin inhibited the protein expression of cyclin B1, cell division cycle 25C (Cdc25C) and Cdc2, but enhanced checkpoint kinase 2 (Chk2), Cdc25C and Cdc2 phosphorylation. In addition, tubulin depolymerization participated in the isorhamnetin-induced cell cycle arrest in G2/M phase. In conclusion, the present results indicated that the anti-proliferative action of isorhamnetin is associated with arrest of the cell cycle in G2/M phase, which is a consequence of activation of the ataxia telangiectasia mutated Chk2 pathway and disruption of microtubule function.
Cervical cancer is the third most common type of cancer among women worldwide, and the majority of cases are associated with infection caused by human papillomavirus (HPV) (
Numerous types of naturally derived phytochemicals have been demonstrated to have anti-cancer effects on cervical cancer cell lines (
Isorhamnetin, an O-methylated flavonol, is mainly extracted from sea buckthorn (
Isorhamnetin was from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). It was first dissolved in dimethyl sulfoxide (DMSO) to generate a stock solution. For cell treatments, the stock solution was further diluted in culture medium as required. The final concentration of DMSO in the culture medium was <0.4% (v/v).
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), propidium iodide and trypsin-EDTA were from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Bovine serum albumin (BSA), DMSO and Trypan blue were from Sigma-Aldrich (Merck KGaA). Penicillin and streptomycin were obtained from M&C Gene Technology (Beijing, China).
The primary antibodies to checkpoint kinase (Chk) 1 (cat. no. 2360), Chk2 (cat. no. 3440) and call division cycle (Cdc) 2 (cat. no. 9116), Cdc25C (cat. no. 4688), phosphorylated (p)-Cdc2 (Tyr15; cat. no. 4539), p-Chk1 (Ser345; cat. no. 2348), p-Chk2 (Thr68; cat. no. 2197) and p-Cdc25C (Ser216; cat. no. 4901), β-actin (cat. no. 4970) (all dilution 1:1,000) and cyclin B1 (cat. no. 4135; dilution 1:2,000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies for α-tubulin (cat. no. ab7750) and β-tubulin (cat. no. ab70187) were obtained from Abcam (Cambridge, MA, USA) and used at dilution 1:500. The secondary antibodies for goat anti-rabbit (cat. no. A0208) and goat anti-mouse (cat. no. A0216) were purchased from Beyotime Institute of Biotechnology (Haimen, China) and used at dilution 1:1,000. For western blots, the antibodies were diluted in 0.5% blocking buffer (add 5 g BSA, 1.22 g Tris and 8.78 g NaCl to 1 l distilled water and adjust pH to 7.5).
HeLa cells were obtained from Bioleaf Company (Shanghai, China) and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were maintained in a humidified atmosphere of 5% CO2 at 37°C and were passaged every 2–3 days.
The anti-proliferative activity of isorhamnetin was measured using a Trypan blue dye exclusion assay (
The cell cycle distribution was assessed by flow cytometry (
HeLa cells were treated with different concentrations of isorhamnetin for 24 h. Subsequently, the cells were washed with PBS. For the extraction of total protein, cells were collected by scraping and incubated for 1 h in sample buffer [150 mmol/l sodium chloride, 20 mmol/l Tris (pH 7.5), 1 mmol/l EDTA, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1% Triton X-100, 1 mmol/l sodium orthovanadate, 1 mmol/l phenyl methane sulfonyl fluoride, 0.5% sodium deoxycholate, 1% protease inhibitor and 20 mmol/l sodium fluoride], and then centrifuged at 12,000 × g for 30 min at 4°C. The total protein concentration in the clear supernatant was evaluated using the Bradford method. Aliquots containing 30 µg protein were subjected to 15% SDS-PAGE. The proteins were then electrophoretically transferred to a nitrocellulose membrane (cat. no. HATF00010; EMD Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at room temperature with a 3% blocking buffer (add 30 g BSA in Tris-buffered saline (TBS) buffer to final volume 1 l). The membranes were rinsed with TBS buffer (add 1.22 g Tris and 8.78 g NaCl to 1 l distilled water and adjust pH to 7.5 with HCl) three times, for 5 min each time. The primary antibodies were added in 10 ml 0.5% blocking buffer and incubated for 2 h at room temperature. The membranes were washed twice for 10 min each time with Tween-20 TBS buffer (add 0.5 ml 0.05% Tween-20 to 1 l TBS buffer). The secondary antibodies were added in a 5 ml 0.5% blocking buffer and incubated for 1 h at room temperature. The membranes were washed twice for 10 min with Tween-20 TBS buffer. Specific signals were detected by enhanced chemiluminescence (Amersham International; GE Healthcare, Little Chalfont, UK). The densitometry was performed using ImageJ software (National Institute of Health, Bethesda, Maryland, USA) and the density of each band was normalized against β-actin. All experiments were performed in triplicate.
Differences between the control and treatment groups were analyzed by analysis of variance, followed by Duncan's multiple range test. SPSS Statistics version 19.0 was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference. Values are expressed as the mean ± standard deviation of at least three independent experiments.
The cell proliferation assay demonstrated that isorhamnetin inhibited the proliferation of HeLa cells at concentrations of >10 µmol/l in a dose-dependent manner (
Cell cycle distribution analysis revealed that treatment with isorhamnetin for 24 and 72 h significantly increased the proportion of cells in G2/M phase (
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A slight increase in the protein level of p-Cdc2 (Tyr15) was observed after isorhamnetin treatment (
Maintenance of a critical threshold of tubulin protein is essential for the function of microtubule networks. Analysis of tubulin protein levels indicated that isorhamnetin downregulated the expression of α-tubulin in a dose-dependent manner, but did not affect the expression of β-tubulin (
The occurrence and progression of cervical carcinoma is associated with HPV infection as well as a wide range of cellular, epigenetic, genetic, immunological and environmental factors (
Initially, the anti-proliferative effect of isorhamnetin on HeLa cells was assessed by the mitochondrial respiration-dependent MTT reduction method. However, as isorhamnetin has light absorption properties, it interfered with the result of the MTT assay. Therefore, the trypan blue exclusion method was used. The results indicated that isorhamnetin inhibited the proliferation of HeLa cells in a dose-dependent manner at 24, 48 and 72 h. Of note, the IC50 of isorhamnetin after 48 h of treatment was nearly three times greater than that after 24 h of treatment. As an underlying cause, it is possible that a repair system was activated in cells treated with isorhamnetin for 48 h, which will be investigated in further experiments.
The cell cycle determines cell proliferation and regulates complex processes that determine cell growth and division. All signaling pathways affecting the cell cycle must be precisely regulated in order to determine the fate of the cell. In almost all cancer cell types, a number of different mechanisms influence the normal cell cycle (
Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins are kinases that are activated in the presence of cell damage signals, and induce either cell cycle arrest or apoptosis. Certain antineoplastic drugs have been demonstrated to activate these two checkpoint proteins (
Activation of Chk1 or Chk2 leads to the phosphorylation of a variety of cell cycle regulatory proteins, including Cdc25C (
Cdc2 [also known as cyclin D kinase 1] and cyclin B1 constitute a complex called mitosis-promoting factor. This complex is involved in regulating G2/M transition (
Microtubules are a component of the cytoskeleton. A large and diverse group of anticancer drugs derived from natural products target microtubules in cancer cells. Microtubules are composed of tubulin proteins (α-tubulin and β-tubulin), and are assembled dynamically through polymerization and de-polymerization. The normal regulation of microtubule assembly serves a key function in cells during M phase (
In conclusion, the present study demonstrated that isorhamnetin significantly inhibits the proliferation of HeLa cells
Not applicable.
The present study was supported by the Projects of International Cooperation and Exchanges of the Ministry of Science and Technology in China (grant no. 2014DFR31230) and the National Natural Science Foundation of China (grant no. 31360382).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JW conceived and designed the study, performed the majority of the experiments and wrote the paper. HS performed the flow cytometric analysis. YB helped to design the study, reviewed and edited the manuscript and approved the final version to be published. JL performed the cell culture. LF helped to analyze the data. WS helped to analyze the data and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Isorhamnetin inhibits the proliferation of HeLa cells. Cells were exposed to different doses of isorhamnetin (1–1,000 µmol/l) for 24, 48 and 72 h. *P<0.05 and **P<0.01 vs. the control group. Values are expressed as the mean ± standard deviation of six independent experiments.
Isorhamnetin arrests HeLa cells at G2/M phase. Flow cytometry was performed to assess the cell cycle distribution of HeLa cells treated with (A-a) 0, (A-b) 10, (A-c) 50 or (A-d) 100 μmol/l isorhamnetin for 24 h, or (B-a) 0, (B-b) 10, (B-c) 50 or (B-d) 100 μmol/l isorhamnetin for 72 h. The percentage of cells in G1, G2/M or S phase was assessed after treatment with various concentrations of isorhamnetin for (C) 24 and (D) 72 h. *P<0.05 and **P<0.01 vs. the control group. Values are expressed as the mean ± standard deviation of triplicate results.
Effect of isorhamnetin on the levels of Chk1, p-Chk1 (Ser345), Chk2 and p-Chk2 (Thr68). The cells were treated with isorhamnetin (10, 50 or 100 µmol/l) for 24 h. (A) Protein levels were evaluated by western blot analysis. (B-E) Quantification of western blot results for (B) Chk1, (C) p-Chk1 (Ser345), (D) Chk2 and (E) p-Chk2 (Thr68). Protein levels were normalized to β-actin. The relative level of protein in the control group was set at 100%. **P<0.01 vs. the control group. Values are expressed as the mean ± standard deviation of triplicate results. p-Chk, phosphorylated checkpoint kinase.
Effect of isorhamnetin on the levels of Cdc25C and p-Cdc25C (Ser216). (A) Protein levels were evaluated by western blot analysis. (B and C) Quantification of western blot results for (B) Cdc25C and (C) p-Cdc25C (Ser216). Protein levels were normalized to β-actin. The relative level of protein in the control group was set at 100%. The cells were treated with isorhamnetin (10, 50 or 100 µmol/l) for 24 h. *P<0.05 and **P<0.01 vs. the control group. Values are expressed as the mean ± standard deviation of triplicate results. p-Cdc, phosphorylated cell division cycle.
Effect of isorhamnetin on the levels of Cdc2, cyclin B1 and p-Cdc2 (Tyr15). (A) Protein levels were evaluated by western blot analysis. (B-D) Quantification of western blot results for (B) Cdc2, (C) cyclin B1 and (D) p-Cdc2. Protein expression was normalized against β-actin. The relative expression of protein in the control group was set at 100%. The cells were treated with isorhamnetin (10, 50 or 100 µmol/l) for 24 h. *P<0.05 and **P<0.01 vs. the control group. Values are expressed as the mean ± standard deviation of triplicate results. p-Cdc, phosphorylated cell division cycle.
Effect of isorhamnetin on the expression of α-tubulin and β-tubulin. The cells were treated with isorhamnetin (10, 50 or 100 µmol/l) for 24 h. (A) Protein expression was evaluated by western blot analysis. Quantification of western blot results for (B) α-tubulin and (C) β-tubulin. Protein levels were normalized to β-actin. The relative level of protein in the control group was set at 100%. *P<0.05 and **P<0.01 vs. the control group. Values are expressed as the mean ± standard deviation of triplicate results.