Ivermectin induces cell cycle arrest and apoptosis of HeLa cells via mitochondrial pathway

Abstract Objectives The aim of study was to investigate the anticancer activities of Ivermectin (IVM) and the possible mechanisms in cells level via cell proliferation inhibition, apoptosis and migration inhibition in model cancer cell HeLa. Materials and methods The MTT assay was used to study the inhibitory effect of IVM on the proliferation of Hela cells, and the cell cycle was analysed by flow cytometry. The neutral comet assay was used to study the DNA damage. The presence of apoptosis was confirmed by DAPI nuclear staining and flow cytometry. Changes in mitochondrial membrane potential and reactive oxygen species (ROS) levels were determined using Rhodamine 123 staining and DCFH‐DA staining. Western blot analysis for apoptosis‐related proteins was carried out. We use scratch test to analyse the antimigration potential of IVM. Results Ivermectin can inhibit the viability of HeLa cells significantly. In addition, treatment with IVM resulted in cell cycle arrest at the G1/S phase which partly account for the suppressed proliferation. Typical apoptosis morphological changes were shown in IVM treatment cells including DNA fragmentation and chromatin condensation. At the same time, the results of flow cytometry analysis showed that the number of apoptotic cells increased significantly with the increase of IVM concentration. Moreover, we observed that the mitochondrial membrane potential collapses and the ratio of Bax/Bcl‐2 in the cytoplasm increases, which induces cytochrome c release from the mitochondria to the cytoplasm, activates caspase‐9/‐3 and finally induces apoptosis. We also found that IVM can significantly increase intracellular ROS content. At the same time, we determined that IVM can significantly inhibit the migration of HeLa cells. Conclusions Our experimental results show that IVM might be a new potential anticancer drug for therapy of human cancer.

It is believed that IVMacts on gamma-amino butyric acid (GABA) receptor and glutamate-gated chloride channels. 3 Due to the low affinity for other mammalian ligand-gated channels, IVM hardly crosses the blood-brain barrier. Consequently, IVM has been generally supposed to be of low toxicity for the health of adults and approved for use in humans worldwide. 4 For example, IVM was widely used for the treatment of river blindness in the form of a free drug, saving countless people's eyesight. 5 The discoverers of IVM were awarded the 2015 Nobel Prize in Physiology or Medicine due to it has a very successful therapeutic effect on many parasitic diseases. 6 In the recent years, IVM has been reported to exert anticancer activities. However, the specific roles of IVM in cancer remain unclear.
Cancer is a group of diseases with characteristics involving abnormal and uncontrolled cell growth, the potential for penetration and diffusion to other parts of body, considered as the leading cause of death in the world. 7 Therefore, the research of antitumour drugs with high efficiency and low toxicity has been the focus of drug discovery. 8 The HeLa cell line derived from cervical cancer cells was the first successful attempt to immortalize human-derived cells in vitro and has served a role analogous to that of a model organism due to its robust growth and unrestricted distribution. 9 Cervical cancer is one of the most common malignancy throughout the world which has become the second female malignant tumour, and has a serious threat to the health and safety of women. 10 Based on these two points above, we chose to use HeLa cells for our study.
Ivermectin has been demonstrated to have anticancer activities on various types of cancer including glioblastoma, chronic myelogenous leukaemia and breast cancer. [11][12][13] Because of the lack of systematic research on the anticancer activity of IVM in HeLa cells, the aim of this study was to investigate the activity and underlying mechanism of IVM against HeLa cells, and to provide experimental basis and theoretical foundation for further research on theory and clinical study.

| Cell culture
HeLa cell line (Human cervical carcinoma) was acquired from ATCC and grown in DMEM medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Hyclone) in an incubator with a humidified air atmosphere of 5% CO 2 at 37°C.

| Cell viability assay
The MTT colorimetric method is a method that can quickly quantify the number and the value-added state of living cells. 14 It is widely used for cytotoxicity detection due to its advantages such as small workload, easy operation, rapidity and good repeatability. 15 The HeLa cells (5 × 10 3 cells/well) were seeded onto 96-well plates. After 24 hours of incubation, cells were treated with the corresponding concentration (0, 2.5, 5, 7.5, 10 and 20 μmol/L) of IVM for 24 and 48 hours. Complete DMEM medium was used as a blank. Four hours prior to the assay, MTT reagent (20 μL/well, 5 mg/mL) was added to cells. We replaced the medium with DMSO (200 μL/well), and then we read the absorbances at 492 and 630 nm by a microplate reader (Bio-Teck, Winooski, VT, USA).

| Cell cycle analysis
The HeLa cells (1 × 10 6 cells/well) were seeded onto 9.6 cm 2 plates overnight and treated with specified concentrations (0, 2.5, 5, 10 and 20 μmol/L) of IVM. Then, the cells were harvested and resuspended in PBS and fixed with 70% ethanol and left at − 20°C overnight. After 12 hours of fixation, cells were centrifuged at 600 g for 5 minutes, washed and resuspended in PBS (200 μL). Then, we added 100 μL propidium iodide solution (500 μg/mL) to the cells and incubated for 15 minutes in the dark. 16 The cell cycle was then analysed by flow cytometry (BD FACS Calibur). Data were analysed by the Flowjo software.

| DNA damage assay
The neutral comet assay was used to study the DNA damage in-

| Mitochondrial membrane potential (ΔΨm) analysis
The effect of IVM on mitochondrial membrane potential was assessed using fluorescence microscopy. HeLa cells were seeded (1 × 10 5 cells/ well) in a 12-well plate and incubated overnight. After HeLa cells were treated with 0, 2.5, 5, 10 and 20 μmol/L IVM for 6 hours, cells were stained with Rhodamine123 for 30 minutes at 37°C. The phenomenon of cells was photographed by fluorescence microscopy. The images were measured using Imagej software.

| Western blotting
After treating HeLa cells (1 × 10 6 cells/well) with 0, 2.5, 5, 10 and 20 μmol/L IVM for 6 hours, the cells were collected, washed with PBS and lysed with RIPA buffer. Protein quantification was performed using BCA Protein Assay Kit. Equal amounts of protein were loaded onto an SDS-PAGE gel for electrophoresis and transferred to polypropylene difluoride (PVDF) membranes. After blocking with 5% nonfat milk for 2 hours, the membranes were incubated with the primary antibody for 12-16 hours. The membranes were then incubated with secondary antibody for 2 hours at room temperature. And the change in target protein expression was detected by a chemiluminescent gel imaging system (Tanon, Shanghai, China).

| Intracellular ROS measurement
The effect of IVM on intracellular reactive oxygen species was evaluated by fluorescence microscopy. 18 HeLa cells were seeded (1 × 10 5 cells/well) in a 12-well plate and incubated overnight. After HeLa cells were treated with 0, 2.5, 5, 10 and 20 μmol/L IVM for 6 hours, cells were tinted with oxidation of DCFH-DA for 30 minutes at 37°C.
The phenomenon of cells was photographed by fluorescence microscopy. The images were measured using Imagej software.

| Antimigration assay
We use scratch test to analyse the antimigration potential of IVM.
HeLa cells (1 × 10 5 cells/well) were seeded in 6-well plate. When the cell density reaches about 70%, we use a 200 μl yellow pipette tip to draw a straight line in the cell culture plate. 19 Since antimetastatic agents usually require long-term use by patients, they should have low cytotoxicity. Therefore, in the current study, we have used IVM at lower concentrations than that used for other assays (2.5, 5, 10, 15 μmol/L). After scratching, HeLa cells were exposed to different concentrations of IVM for 48 hours. The images of cell migration were captured using fluorescence microscopy. The data analysis was performed using Imagej soft-ware.

| Data analyses
At least three independent biological replicates were performed in all experiments. Data were shown as mean ± standard deviation. To determine differences between groups, we performed the Dunnett test using ANOVA (P < 0.05 indicates statistical significance). SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.

| Inhibition of HeLa cells viability by IVM
To evaluate the cytotoxic of IVM on HeLa cells, we analysed the

| IVM-induced DNA damage in HeLa cells
We used single-cell gel electrophoresis (comet assay) to evaluate the DNA damage induced by IVM. During electrophoresis, the broken DNA fragments move towards the positive electrode by carrying a negative charge, and the unbroken DNA does not move. After staining, the nucleus forms a bright head, and the DNA fragments form a tail, which resembles a comet-like shape. The more severe the DNA damage, the longer the tail length. It can be seen from the Figure 3A that the nuclear chromatin DNA of the untreated control group of IVM is basically not broken, the nucleus is intact, and it is round, and no obvious comet-like tail is observed. However, the nuclear chromatin DNA of the treatment group is obviously broken and formed a long comet-like tail, the "tailing" phenomenon. The percentages of comet-positive cells of HeLa increased significantly after treated by IVM in a dose-dependent way ( Figure 3B). The distributions of IVMtreatment HeLa cells, with respect to comet assay parameters including tail lengths and tail DNA are presented in Table 2 (**P < 0.01).
The results showed that there was no obvious DNA damage in the untreated control group, and the DNA damage of HeLa cells was significantly improved after IVM treatment.

| IVM-induced apoptosis in HeLa cells
After treatment with different concentrations of IVM for 24 hours, IVM-induced apoptosis was measured by flow cytometry ( Figure 4A). The different labelling patterns in the annexin V/PI analysis were used to identify the different cell populations where Small alphabet indicated statistically significant differences between two groups in the same column (P < 0.05).
TA B L E 1 The half maximal inhibitory concentration (IC 50 ) of HeLa cells exposed to IVM

| IVM-induced mitochondrial membrane potential collapse in HeLa cells
Mitochondrial membrane potential (MMP) is one of the important indicators for maintaining normal mitochondrial configuration and function. 20 Decreased MMP is one of the important factors leading to apoptosis and is considered to be the first step of the apoptosis cascade. 21 Therefore, we tried to evaluate the influence of IVM on MMP in HeLa cells. We found that exposure of HeLa cells to different concentrations (0, 2.5, 5, 10 and 20 μmol/L) of IVM for 6 hours resulted in a gradual decrease in green fluorescence intensity ( Figure 5A,B). The study shows that the decrease of MMP was brought about by IVM treatment in HeLa cells (**P < 0.01).

| Effect of IVM on the expression of apoptosisrelated proteins in HeLa cells
The role of mitochondria in the mechanism of apoptosis has at-

| Effect of IVM on caspase-3, caspase-9,P53 and PARP in HeLa cells
The process of apoptosis involves a complex cascade of proteolytic hydrolysis. 22 The Caspase family plays an extremely important role in the process of apoptosis. 23 P53 is a tumour suppressor protein that plays an important role in the mitochondrial apoptosis pathway. 24 To further investigate the apoptosis of HeLa cells caused by IVM, we examined the activity of caspase-3, caspase-9, p53 and PARP by Western blot assay. As shown in Figure 7, caspase-3 and  The data are shown as the means ± SD of three independent experiments. Small alphabet indicated statistically significant differences between any two groups in the same column (P < 0.05).
caspase-9 were both activated while cleaved PARP increased and P53 protein expression was significantly increased.

| IVM-induced generation of intracellular ROS in HeLa cells
We

| Migration of HeLa Cells was Inhibited by IVM
We further verified the inhibitory effect of IVM on HeLa cell migration using antimigration assay ( Figure 9A). Since higher concentrations of IVM may cause apoptosis, this study was performed at lower concentrations to confirm the antimigratory potential. After

| D ISCUSS I ON
In recent years, the use of bacterial or fungal fermentation to prevent and treat cancer is attracting more and more attention. 25 IVM is a semi-synthetic macrocyclic lactone derivative of the avermectin family. 26 Nowadays, because of it has no side effects on human safety, IVM has been approved for use in humans to treat on chocerciasis and lymphatic filariasis. 27 Accumulating evidence suggests that IVM has anticancer activities against breast cancer, ovarian cancer, neurofibromatosis and chronic myeloid leukaemia. In our study, ing in cell cycle arrest. 28 Cell cycle progression is one of a pivotal signalling mechanisms of homoeostasis maintenance in healthy tissues and normal cells. 29 Therefore, induction of cell arrest in cancer cells is one of the useful strategies for anticancer drug development. 30 Cell cycle analysis shows that IVM induces G1/S cycle arrest in HeLa cells, which means that IVM inhibits proliferation of HeLa cells via cell cycle arrest resulting from DNA damage.
It is well known that three pathways can lead to cell death, namely apoptosis, autophagy and cell necrosis. 31   As we all know, the apoptosis pathway is divided into two major types: intrinsic pathway (mediated by mitochondria) and the extrinsic pathway (mediated by death receptors). 33 In order to identify the type of IVM-induced apoptosis ef- Rapid metastasis is one of the biggest malignant features of cancer cells. 35 The invasion and metastasis of malignant tumours are a series of complex and multi-step interactions. 36 It involves the malignant proliferation of cancer cells, the adhesion of tumour cells, movement, invasiveness and the ability to metastasize. Tumour invasion is the premise of cancer metastasis. 37 Invasion does not mean that metastasis must occur. However, tumour metastasis must invade. 38 We determined that IVM can significantly inhibit the migration of HeLa cells by the scratch test.
In conclusion, we have demonstrated that IVM can significantly inhibit the proliferation of HeLa cells via cell cycle arrest resulting from DNA damage, and induced apoptosis of HeLa cells through the mitochondrial pathway. At the same time, we found that IVM can inhibit its migration activity significantly. Through these studies, we determined that IVM has significant anticancer activity against HeLa cells. However, the study of the effect of IVM on cancer in vivo has not yet been carried out. This will be the focus of our future work.