The human HeLa and SiHa cervical cancer cell lines, and the non-cancerous 293 and CCD-1095Sk cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in 37°C with 5% CO₂ according to the instructions provided by ATCC. Icaritin was purchased from Yuanye Biotechnology (Shanghai, China). The purity was measured by high-performance liquid chromatography (15) and determined to be 99.6%. Stock solutions of icaritin were prepared in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and working solutions were in complete cell culture medium. Vehicle control samples included the same amount of DMSO in the absence of icaritin. N-acetyl cysteine (NAC) was purchased from Calbiochem (EMD Millipore, Billerica, MA, USA). The sources of additional reagents were specified in the relevant sections.

The cells were seeded in 96-well plates at a density of 2,000 cells per well for 12 h, and then treated with icaritin or vehicle control for 24 or 48 h. A total of 20 µl MTT (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) solution (5 mg/ml in PBS) was added to each well. The plate was incubated at 37°C for an additional 4 h. Following removal of the culture medium, the cells were incubated in 150 µl 100% DMSO on a plate shaker for 10 min. The absorbance was measured at 595 nm was measured by a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The data were analyzed using the GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA).

The cells were seeded in 6-well plates at 2,000 cells/well for 12 h and then treated with different concentrations of icaritin or vehicle control for 8 days. Following washing with PBS, the cells were fixed in ice-cold methanol and briefly stained with crystal violet solution (0.5% in 25% methanol; Sigma-Aldrich; Merck KGaA). The plates were air-dried overnight and photographs were captured with a Canon digital camera (PowerShot ELPH 180 20-Megapixel; Canon, Inc., Tokyo, Japan). Ethanol (70%) was added to dissolve the violet crystals and the absorbance at 595 nm was measured by a microplate reader (Bio-Rad Laboratories, Inc.). The data were analyzed using the GraphPad Prism software.

The cells were seeded in 12-well plates at a density of 3×10⁵ cells/well until they reached 70–80% confluence as a monolayer. A vertical straight scratch across the center of each well was made by gently and slowly scratching the monolayer with a 1 ml pipette tip in one direction. The wells were washed twice with medium to remove the detached cells and fresh medium was added that contained different concentrations of icaritin and/or vehicle control. phase-contrast microscopy images were captured at 0, 24 and 48 h and analyzed using the ImageJ software (National Institutes of Health, Bethesda, MA, USA).

Total cellular ROS were measured by flow cytometry using a cell-based ROS assay kit (Beyotime Institute of Biotechnology, Haimen, China). Following drug treatment, the cells were washed twice with PBS and subsequently incubated with 10 µM dichlorofluorescin diacetate (DCFH-DA) at 37°C for 30 min in the dark. The cells were collected following trypsin digestion (Thermo Fisher Scientific, Inc.) and then analyzed by the FACSCaliber flow cytometer (BD Biosciences, San Jose, CA, USA). Total cellular ROS levels were expressed as the averaged DCF fluorescence intensity of the cells. The data presented is average of three independent experiments.

Following drug treatment, the cells were washed once by PBS, fixed in methanol/acetone (v/v, 1/1) at 4°C for 15 min, and washed 3 times with PBS. Fixed cells were blocked in 0.3% Triton X-100 with 1.5% bovine serum albumin (BSA) at room temperature for 30 min, incubated with rabbit-anti-TP53-binding protein 1 (53BP1; 1:1,000; cat. no. A300-272A; Bethyl Laboratories, Inc., Montgomery, TX, USA) and/or rabbit-anti-γH2AX histone (1:500; cat. no. bs-3185R; BIOSS, Beijing, China) primary antibodies at 4°C overnight, followed by incubation with Cy3-conjugated goat-anti-rabbit (1:250; cat. no. 111-165-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) secondary antibody for 1 h at room temperature. The slides were washed three times with PBS and sealed in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA).

The comet assay was performed using the OxiSelect Comet assay kit (Cell Biolabs, Inc., San Diego, CA, USA) following the manufacturer's instructions (40). The cells were collected by trypsin digestion, washed twice with PBS, and then resuspended in 1.2% low-melting point agarose maintained at 37°C. The density of the cells used was 1×10⁵ cells/ml. A total of 80 µl of cell-containing agarose was layered on a frosted glass slide. The slide was transferred to 4°C for 15 min and submerged in pre-cooled lysis buffer (100 mM EDTA, 2.5 M NaCl, 10 mM Tris-HCl, 1% Triton X-100 and 10% DMSO, pH 10.0) at 4°C for 1.5 h in the dark. The slides were washed twice in enzyme buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA and 0.2 mg/ml BSA, pH 8.0) and then incubated in enzyme buffer with or without 8-oxoguanine glycosylase (OGG1; 1.0 µg/ml; OriGene Technologies, Inc., Rockville, MD, USA) at 37°C for 45 min. Following brief washing with enzyme buffer, the slides were denatured in pre-cooled alkaline buffer (300 mM NaOH, 1 mM EDTA) in an electrophoresis chamber for 30 min and then electrophoresis was performed under 300 mA and 50 V for 30 min. The slides were incubated in cooled neutralizing buffer (250 mM Tris-HCl, pH 7.5) for 30 min, immersed in cold 70% ethanol for 5 min and then dried in air. The cells on the slides were stained with Vista Green DNA dye at room temperature for 15 min in the dark. The images were captured using an Olympus fluorescent microscope (Olympus Corporation, Tokyo, Japan). The tail moment was defined as percentage of tail DNA × tail length, quantified using the CASP software (Perceptive Instruments, Ltd., Edmunds, UK).

The cells were washed twice with PBS and removed from the plate using a scraper and 100 µl radioimmunoprecipitation buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and 50 mM Tris, pH 8.0) containing 1 mM phenylmethane sulfo-nylfluoride (Santa Cruz Technology, Inc., Dallas, TX, USA).

Following centrifugation at 12,000 × g for 20 min at 4°C, the protein concentration levels in the supernatants were measured using a bicinchoninic acid kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). The samples were denatured at 95°C for 10 min, separated on a 12% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (EMD Millipore). The membranes were blocked in Tris-buffered saline with Tween-20 [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween-20; PBST) containing 5% (w/v) nonfat milk for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C [B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. sc-7382) and apoptosis regulator Bax (Bax; 1:500; cat. no. sc-7480) from Santa Cruz Biotechnology, Inc.; cleaved caspase 3 (1:500; cat. no. ab2302), cleaved caspase 9 (1:200; cat. no. ab2324) and X-linked inhibitor of apoptosis protein (XIAP; 1:1,000; cat. no. ab28151) from Abcam (Cambridge, UK); cyclin-dependent kinase 1 (CDK1)-pT14 (1:1,000; cat. no. bs-3091R), cell division cycle 25C (CDC25C)-pS216 (1:1,000; cat. no. bs-3096R), β-actin (1:5,000; cat. no. bs-0061R) and matrix metalloproteinase 9 (MMP9; 1:1,000; cat. no. bs-4593R) from BIOSS]. Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated goat-anti-rabbit (1:10,000; cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc.) secondary antibody for 1 h at room temperature. The signals were visualized with an ECL kit (EasySee Western Blot kit; Beijing Transgen Biotech Co., Ltd., Beijing, China).

Following drug treatment, the cells were collected by trypsin digestion, washed twice with PBS, and fixed in 70% alcohol at −20°C for 1 h. Fixed cells were resuspended in 300 ml of freshly prepared PBS with 0.1% Triton X-100, 0.2 mg/ml DNase-free RNase A (Sigma-Aldrich; Merck KGaA), 10 µg/ml propidium iodide (Roche Diagnostics, Basel, Switzerland), and incubated at 37°C for 20 min in the dark. The cells were filtered through a Filcon nylon mesh (BD Biosciences) and analyzed using a FACScalibur flow cytometer and ModFit software (BD Biosciences).

Following drug treatment, cells were collected by trypsin digestion, washed twice with PBS and stained in working solution using an Annexin V-fluorescein isothiocyanate/PI dual staining kit (BestBio Science, Shanghai, China) according to the manufacturer's instructions. The stained cells were analyzed by the FACScalibur flow cytometer and the CellQuest software (BD Biosciences).

The mitochondrial membrane potential was measured using the JC-1 dye (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The cells were washed twice with PBS, incubated with 1 ml JC-1 working solution for 20 min at 37°C, and sealed in VECTASHIELD medium (Vector Laboratories). Cell images were imaged with an Olympus fluorescent microscope.

The data are expressed as the mean ± standard deviation and the results were representative of three independent experiments. All statistical analyses were performed using the GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). The statistical comparisons between two groups were performed by unpaired two-tailed Student's t-test and multigroup comparisons were conducted by using two-way ANOVA followed by the Dunnett or Tukey test. P<0.05 was considered to indicate a statistically significant difference.

It has been reported that icaritin (Fig. 1A) induces considerable cytotoxicity against several different types of cancer cells by mechanisms that are not yet fully understood (26–34). To examine the effects of icaritin on cervical cancer, the cytotoxicity of icaritin against human HeLa and SiHa cells was analyzed. HeLa and SiHa cancer cells were treated with a concentration range of icaritin for 24, 48 and/or 72 h. The results of the MTT assay indicated that the proliferation rates of HeLa and SiHa cancer cells were significantly suppressed by icaritin (Fig. 1B-D). Following 48 h of treatment, the half maximal inhibitory concentration (IC₅₀) values of icaritin for HeLa and SiHa cells were 12.5 and 17 µM, respectively (Fig. 1B). The viabilities of HeLa and SiHa cells were reduced to 47.5 and 60%, respectively, following treatment by 12.5 µM icaritin for 48 h, while those of the non-cancerous CCD-1095Sk fibroblasts and H293 epithelial cells were not changed (Fig. 1C). These results indicated that icaritin was selectively more toxic to cancer cells. Overall, the inhibition of cancer cell proliferation by icaritin exhibited optimal dose- and time-dependency (Fig. 1D).

Subsequently, a colony formation assay was used to confirm the inhibitory activity of icaritin against HeLa and SiHa cervical cancer cells. After 8 days of treatment with icaritin at the IC₅₀ determined in the cytotoxicity assay, fewer and smaller colonies were observed for SiHa and HeLa cells compared with 0 µM treatment (Fig. 2A and B). In contrast to this dose treatment, higher concentration levels of icaritin (equivalent to 2× IC₅₀) resulted in further inhibition of cellular proliferation (Fig. 2A and B). However, the growth curves of the non-cancerous CCD-1095Sk fibroblasts and 293 epithelial cells that were treated with the same concentration points were much less affected (data not shown). Taken collectively, these results suggested that icaritin dose-dependently and selectively suppressed the growth of HeLa and SiHa cervical cancer cells.

In addition to these proliferation and survival assays, the impact of icaritin on the growth and/or migration of HeLa and SiHa cervical cancer cells was further analyzed in wound healing assays. The growth and/or migration of both HeLa and SiHa cells were significantly decreased in the presence of icaritin (Fig. 2C and D). Given the effects revealed by the MTT and colony formation assays above, the decreased wound healing was likely a result of an inhibitory effect on proliferation, rather than on migration.

Icaritin has been reported to stimulate ROS generation in specific cell types (34–38). To determine whether icaritin could induce ROS generation in HeLa and SiHa cervical cancer cells, the fluorescent probe DCFH-DA was used to measure total cellular ROS. Treatment with icaritin at concentrations equivalent to the IC₅₀ values for 12 h caused a significant increase in ROS levels in HeLa and SiHa cancer cells, which was blocked by pre-treatment with 5 mM NAC (Fig. 3A and B). A significant increase in ROS levels was noted as early as 3 h after initiation of icaritin treatment (Fig. 3C), whereas a longer treatment did not result in an additional increase in ROS levels. Notably, the inhibition of icaritin-induced ROS increase by NAC significantly reduced the cytotoxicity of icaritin against HeLa and SiHa cells (Fig. 3D). These results suggested that treatment of cervical cancer cells with icaritin caused a rapid and robust increase in ROS levels, which contributed to the suppression of cancer cell proliferation by icaritin.

Elevated ROS levels results in oxidative stress, which can damage cellular components and affect cell survival. For example, repair of ROS-oxidized deoxynucleotides in DNA, such as 8-oxo-2′-deoxyguanosine (8-oxo-dG), may result in accumulation of repair-associated DNA strand breaks, and subsequent induction of cell cycle arrest and programmed cell death (41). To gain insight into the physiological implications of the icaritin-induced ROS overload in cancer cells, the number of DNA strand breaks prior to and following treatment with icaritin was compared using the comet assay (single-cell gel electrophoresis). Alkaline conditions were used to convert all single-strand DNA breaks (SSB) to double-strand breaks (DSB), suggesting that the size and intensity of the comet tail (tail moment) was proportional to the number of total DNA strand breaks (SSBs + DSBs). The results indicated that after 24 h incubation of HeLa and SiHa cancer cells with 1× IC₅₀ icaritin (12.5 µM for HeLa, 17 µM for SiHa), the number of cancer cells with a comet tail, and the size and intensity of the comet tail (tail moment), were increased (Fig. 4A and B). Treatment with 2× IC₅₀ of icaritin (25 µM for HeLa, 34 µM for SiHa) further increased the number of comet tail-containing cells and their corresponding tail size (Fig. 4A). These effects were blocked by pre-treatment with the ROS inhibitor NAC, indicating that the DNA strand breaks occurred via ROS production (Fig. 4C).

OGG1 is a glycosylase that specifically removes the oxidized guanine base of 8-oxo-dG in DNA, leaving an apurinic site, which can be converted into a DSB during an alkaline comet assay. Thus, incubation of the cells with OGG1 prior to electrophoresis can aid the assessment of the amount of 8-oxo-dG in DNA during the comet assay. The data indicated that incubation of icaritin-treated cells with OGG1 further increased the tail moments (Fig. 4B), suggesting that icaritin-induced ROS overload caused extensive oxidative DNA damage, which subsequently resulted in large numbers of DNA breaks.

Subsequently, immunostaining was used to directly visualize and quantify cellular DSBs by analyzing the DSB markers γH2AX and 53BP1. Treatment with icaritin for 12 h caused a dramatic increase in the number of cells with brightly stained nuclear 53BP1 and γH2AX foci (Fig. 5A-C). The earliest increase in the foci of γH2AX was noted at 3 h post-icaritin treatment, and for 53BP1 it was noted at 6 h post-icaritin treatment in HeLa (Fig. 5B) and SiHa cancer cells (Fig. 5C). Higher concentration of icaritin resulted in a higher number of positive cells and higher number of positive foci per cell. Pre-incubation with 5 mM NAC completely blocked the icaritin-induced increase in 53BP1 and γH2AX staining (data not shown).

To assess the consequences of the icaritin-induced DNA damage, icaritin-induced changes in the cell cycle distribution were analyzed by flow cytometry. The results revealed that icaritin increased the percentage of cells at the G₂/M phase, while slightly decreasing the percentage of cells at the S phase (Fig. 6). Following treatment with 25 µM icaritin for 24 h, the percentage of HeLa cells at the G₂/M phase was increased from 6.23 to 19.43%. A similar effect was noted after treatment SiHa treatment with 34 µM icaritin for 24 h (Fig. 6A). The percentage of SiHa cells at the G₂/M phases was increased from 4.15 to 17.35% (Fig. 6A). When the cells were pre-incubated with NAC, the increase in the percentage of G₂/M cells was partially rescued (Fig. 6A and B). These results indicated that icaritin-induced ROS caused cell cycle arrest and blocked the progression of cervical cancer cells to the G₂/M phases of the cell cycle.

Consistent with the observations of cell cycle analysis, examination of the nuclear morphology of icaritin-treated HeLa cells demonstrated that icaritin dose-dependently increased the number of apoptosis hallmarks, namely pyknosis and condensed chromatin (Fig. 7A). This suggested that apoptosis was induced by icaritin treatment.

The percentage of apoptotic cells was analyzed by Annexin V and propidium (PI) double-staining and flow cytometry. The data demonstrated that icaritin dose- and time-dependently induced apoptosis in HeLa and SiHa cells (Fig. 7B and C). Following treatment with icaritin (1× IC₅₀ and 2× IC₅₀) for 24 h, the percentage of apoptotic HeLa cells increased to 8.67 and 12.35%, respectively, and the percentage of apoptotic SiHa cells increased to 15.61 and 23.16%, respectively (Fig. 7B). Longer treatment (48 h) resulted in higher percentage of apoptotic cells (11.11 and 17.36% for HeLa, 30.05 and 33.11% for SiHa; Fig. 7B and C). Pre-incubation with NAC significantly blocked icaritin-induced apoptosis (Fig. 7C).

The loss of mitochondrial membrane potential is characteristic of the activation of the intrinsic apoptotic pathway. The mitochondria membrane potential probe, JC-1, was used to assess the integrity of mitochondrial membranes in HeLa and SiHa cells. The results demonstrated that 24 h treatment with icaritin dose-dependently increased the intensity of green fluorescence, while the intensity of the red fluorescence decreased (Fig. 7D), suggesting a prompt dissipation of mitochondrial membrane potential and an activation of the intrinsic apoptotic pathway caused by icaritin treatment.

To further explore the mechanism of action of icaritin, western blot analysis was used to analyze the changes of proteins associated with apoptosis. Treatment of the cells with the icaritin IC₅₀ for 24 h decreased MMP-9 levels in HeLa and SiHa cells, whereas NAC blocked the icaritin-induced decrease in MMP-9 (Fig. 8A). This indicated that ROS have a role in the downregulation of MMP-9, which may be involved in inhibited cell migration. The ATR serine/threonine kinase (ATR)-checkpoint kinase 1 (Chk1) axis senses DSBs and replication stress to regulate DNA repair, replication and cell cycle progression and/or induce apoptosis (42). Treatment of the cells with icaritin for 24 h resulted in reduced expression levels of CDK1-pT14 and an increase in the expression levels of CDC25C-pS216 (Fig. 8B). Both changes were eliminated by NAC pre-treatment, indicating that ROS-associated activation of the ATR-Chk1 pathway occurs in icaritin-treated cancer cells, and that inhibition of CDK1 activity was potentially responsible for arresting the cell cycle at the G₂/M phase. Finally, treatment of the cells with icaritin for 24 h dose-dependently increased the levels of the pro-apoptotic protein Bax, and the levels of activated caspase 3 and 9 enzymes; whereas, it concomitantly decreased the levels of the anti-apoptotic proteins, Bcl-2 and XIAP (Fig. 8C and D). These findings suggest that icaritin activated the intrinsic apoptotic pathway, potentially as a result of the DDR signaling pathway.