The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Eupalinolide J induces apoptosis, cell cycle arrest, mitochondrial membrane potential disruption and DNA damage in human prostate cancer cells
Zeqi WuXintong XuLingjie DaiYiqi WangBo YangHuajun ZhaoChenghua Lou
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2020 Volume 45 Issue 1 Pages 15-23

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Abstract

Eupalinolide J (EJ) is a new sesquiterpene lactone isolated from Eupatorium lindleyanum DC. In the present study, we investigated the anti-cancer activity of EJ on cell proliferation in human prostate cancer cells. The MTT results indicated that EJ showed marked anti-proliferative activity in PC-3 and DU-145 cells in a dose- and time-dependent manner. DAPI staining analysis demonstrated that this effect was mediated by induction of cell apoptosis. Flow cytometric analysis indicated a significant increase in apoptotic cells, cell cycle arrest at G0/G1 phase and disruption of mitochondrial membrane potential (MMP) after EJ treatment. Meanwhile, the activation of caspase-3 and caspase-9 was visibly observed. Furthermore, our results demonstrated that the expression levels of γH2AX, p-Chk1 and p-Chk2 were significantly up-regulated, suggesting the induction of DNA damage responses in EJ-treated prostate cancer cells. The above results indicated that EJ exhibited effective anti-cancer activity in vitro. It could be a promising candidate agent for the clinical treatment of prostate cancer.

INTRODUCTION

Prostate cancer is the second most frequently diagnosed cancer and the fifth leading cause of cancer death in men worldwide. The number of patients diagnosed with prostate cancer is increasing every year. It was estimated that there were 1.3 million new cases of prostate cancer and 359,000 associated deaths worldwide in 2018 (Bray et al., 2018). Currently, surgery, radiotherapy and chemotherapy are the traditional strategies for prostate cancer therapy. However, only a few patients benefit from those strategies. The morbidity and mortality rates are still high. Therefore, it is necessary to develop novel effective agents for prostate cancer therapy.

Recently, sesquiterpene lactones in medicinal plants have been attracted much attention for their diverse biological activities, including anti-cancer (Alexandre Schefer et al., 2017), anti-inflammatory (Wu et al., 2018, 2017), and antibacterial effects (Labed et al., 2019). Specifically, their anti-cancer activities have gained increasing attention from many researchers. Extensive studies have been performed to demonstrate their anti-cancer effects and molecular mechanisms. Leptocarpin, a plant-derived sesquiterpene lactone, could trigger programmed cell death by inhibition of NF-κB pathway in human cancer cells (Bosio et al., 2015). Bigelovin suppressed tumor growth through inducing apoptosis and autophagy via inhibition of mTOR pathway in liver cancer (Wang et al., 2018a). Sesquiterpene lactones derived from Saussurea lappa induced apoptosis and inhibited invasion and migration in neuroblastoma cells (Tabata et al., 2015). Therefore, sesquiterpene lactones are a promising source for the development of novel anti-cancer agents.

Eupatorium lindleyanum DC. (Compositae) is a traditional Chinese medicine, which has been widely used to treat cough and tracheitis (Yang et al., 2007). A variety of biological activities of this herb have been identified, including anti-inflammatory (Wang et al., 2018b), anti-cancer (Tian et al., 2018; Yang et al., 2019, 2016) and anti-oxidant (Yan et al., 2011) activities. Eupalinolide J (EJ), one of the main compounds in this plant, is a novel sesquiterpene lactone isolated in our lab. However, the effects of EJ on suppression of prostate cancer cells have not been well evaluated. In this study, the authors investigated the anti-cancer activity of EJ in human prostate cancer cells. Our data demonstrated that EJ inhibited the growth of prostate cancer cells via induction of apoptosis, cell cycle arrest, MMP disruption and DNA damage.

MATERIALS AND METHODS

Reagents

Eupalinolide J (EJ) was isolated from Eupatorium lindleyanum DC as we previous described (Yang et al., 2019). The purity of EJ was above 95%. The structure of EJ is shown in Fig. 1. Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco BRL (Grand Island, NY, USA); 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4’,6-diamidino-2-phenylindole (DAPI) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing (Hangzhou, China), Propidium iodide (PI)/RNase staining kit and Annexin V-FITC/7AAD kit were purchased from Becton Dickinson (San Diego, CA, USA). The mitochondrial membrane potential detection kit was purchased from Beyotime (Shanghai, China). Antibodies against γH2AX (#9718), p-Chk1 (#2348), p-Chk2 (#2197), caspase-9 (#9508), caspase-3 (#9662), cleaved caspase-3 (#9664), cleaved caspase-9 (#9501) and GAPDH (#5174) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Fig. 1

Chemical structure of Eupalinolide J (EJ).

Cell culture

PC-3, DU-145 and MCF-10A cell lines were obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology, China Academy of Sciences (Shanghai, China). Cells were grown in DMEM culture medium supplemented with 10% FBS, penicillin G (100 U/mL) and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Cell viability assay

The MTT assay was employed to assess the anti-cancer activity of EJ in human prostate cancer cells. Cells in exponential growth were harvested and planted into 96-well plates at a density of 5 × 103 cells/well. Then different concentrations of EJ (at final concentrations of 0, 2.5, 5, 10 and 20 μM) were used to treat the cancer cells for 24, 48 and 72 hr. After incubation, 20 μL MTT solution (5 mg/mL) was added and the cells were cultured in the incubator for another 4 hr. The optical density was measured at 570 nm by a microplate reader (BIO-RAD, Hercules, CA, USA).

DAPI staining

Cancer cells were seeded in 12-well plates at a density of 1 × 105 cells/well and treated with or without EJ for 24 hr. After that, cells were fixed, permeabilized and stained with DAPI (10 μg/mL) reagent for 20 min in the dark. Changes in cell nuclear morphology were observed under a fluorescence microscope (Nikon, Japan).

Annexin V/PI double staining assay

Cancer cells were seeded in 6-well plates at a density of 3 × 105 cells/well and treated with different concentrations of EJ for 24 hr. Then, cells were trypsinized, washed with cold PBS and resuspended in 500 µL of 1 × binding buffer. Subsequently, 5 µL of Annexin V-fluorescein isothiocyanate (FITC) and PI were added, respectively. Cells were stained for 15 min at room temperature in dark. Samples were analyzed by flow cytometry (Guava Technologies; Merck KGaA, Darmstadt, Germany) and data were processed with CellQuest software.

Evaluation of mitochondrial membrane potential (MMP)

Cancer cells (3 × 105 cells/well) were seeded in 6-well plates and incubated with different concentrations of EJ. After incubation for 24 hr, cells were harvested, washed twice with cold PBS and stained with JC-1 (10 μg/mL) in dark for 15 min at room temperature. Subsequently, the MMP in prostate cancer cells was measured by flow cytometry (Guava Technologies; Merck KGaA).

Cell cycle analysis

Cells (3 × 105 cells/well) were plated in 6-well plates and treated with various concentrations of EJ for 24 hr. Cells were harvested, washed twice with ice-cold PBS and fixed in 70% ethanol at -20°C overnight. On the second day, cells were washed again with ice-cold PBS and stained with PI/RNase for 15 min at room temperature avoiding light. Cell cycle in prostate cancer cells was detected by flow cytometry (Guava Technologies; Merck KGaA).

Western blotting analysis

Prostate cancer cells were seeded in 10 cm dishes at a density of 6 × 105 cells/well and incubated with different concentrations of EJ for 24 hr. After incubation, the cells were harvested and lysed. The protein concentration was quantified. Equal amounts of proteins were subjected to SDS-PAGE and transferred to PVDF membrane. The membranes were then blocked with 5% nonfat milk and incubated with the primary antibodies overnight at 4°C. Following washing with Tris-buffered saline-5% Tween 20 (TBST) solution, the membranes were incubated with secondary antibody at room temperature for another 2 hr. Protein bands were detected by enhanced chemiluminescence (ELC) (BIO-RAD, USA).

Statistical analysis

Experiments were performed in triplicate. All data are presented as the mean ± standard deviation (SD). Statistical differences between the groups were assessed by one-way ANOVA using SPSS 18.0 software. *P value < 0.05 was defined as statistical significance.

RESULTS

Effects of EJ on prostate cancer cell viability

The cytotoxicity of EJ on prostate cancer cells was determined by MTT assay. Cells were exposed to various concentrations of EJ for 24, 48 and 72 hr. MTT results indicated that EJ showed significant inhibitory effects on the proliferation of PC-3 and DU-145 cells in a dose- and time-dependent manner (Fig. 2A and B). The IC50 values were 2.89 ± 0.28 µM for PC-3 cells and 2.39 ± 0.17 µM for DU-145 cells at 72 hr. Interestingly, EJ did not show significant inhibitory effects on normal MCF-10A breast epithelial cells (Fig. 2C). The inhibitory effects of EJ on prostate cancer cells could be a result of apoptosis induction. Hence, we next examined whether EJ could induce apoptotic cell death in prostate cancer cells.

Fig. 2

Anti-proliferative effects of EJ on prostate cancer cells. (A) Effects of EJ on the growth of PC-3 cells. (B) Effects of EJ on the growth of DU-145 cells. (C) Effects of EJ on the growth of MCF-10A cells. Prostate cancer cells were treated with various doses of EJ for different time points (24, 48 and 72 hr). MCF-10A cells were treated with EJ for 24 hr. After treatment, MTT assay was employed to evaluate the cell viability. Data are presented as Mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Effects of EJ on the nuclear morphology in prostate cancer cells

To further identify whether EJ induced cell apoptosis in prostate cancer cells, we first examined the nuclear morphology in EJ-treated prostate cancer cells by DAPI staining. DAPI staining is widely used to identify apoptotic cell death induced by anti-cancer agents. As shown in Fig. 3, in control groups, the cell nuclei were of regularly shaped, similar sizes and evenly stained. Compared with the control groups, EJ-treated prostate cancer cells showed significant condensed and fragmented chromatin, nuclear body fragments and irregular edges in the nuclei with bright fluorescence. The above morphological changes are characteristic of cell apoptosis. These results indicated that EJ induced cell apoptosis in prostate cancer cells.

Fig. 3

Effects of EJ on the nuclear morphology in prostate cancer cells. Cells were pretreated with EJ for 24 hr. After incubation, cells were stained with DAPI and the nuclear morphology was immediately observed. Photographs were taken using a fluorescence microscopy (× 40).

EJ induced apoptosis in prostate cancer cells

To further identify the effects of EJ on apoptosis induction in prostate cancer cells, Annexin V-FITC/7AAD staining was employed to quantify the number of apoptotic cells in EJ-treated cells by using flow cytometry. As shown in Fig. 4, after incubation with EJ, the number of apoptotic cells was significantly increased. These results demonstrated that EJ suppressed the growth of prostate cancer mainly via apoptosis induction.

Fig. 4

EJ induces cell apoptosis in prostate cancer cells. Cells were treated with EJ for 24 hr. Then treated cells were trypsinized, washed with cold PBS and resuspended in 500 µL of 1 × binding buffer. Subsequently, 5 µL of Annexin V-fluorescein isothiocyanate (FITC) and PI were added. Cells were stained for 15 min at room temperature in dark. Samples were subsequently analyzed by flow cytometer. Quantified histograms display the effects of EJ on cell apoptosis. Data are presented as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

EJ induced the dissipation of MMP in prostate cancer cells

It was well-established that mitochondrial dysfunction is critical for the apoptotic pathway. Loss of MMP is an indicator of intrinsic apoptosis due to the release of pro-apoptotic proteins in the cytosol. In the present study, the MMP detection in cancer cells was using the membrane-permeable JC-1 dye. Compared with the control groups, EJ treatment in PC-3 and DU-145 cells induced the dissipation of MMP in a dose-dependent manner as indicated by the increased green fluorescent intensity (Fig. 5), suggesting loss of MMP in prostate cancer cells.

Fig. 5

EJ induces loss of MMP in prostate cancer cells. Cells were treated with different concentrations of EJ for 24 hr. After incubation, cells were harvested, washed twice with cold PBS and stained with JC-1 (10 μg/mL) in dark for 15 min at room temperature. Subsequently, the MMP in prostate cancer cells was measured by flow cytometry. Data are presented as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001.

EJ induced cell cycle arrest in prostate cancer cells

To further examine whether the inhibitory effects of EJ on cell growth were associated with induction of cell cycle arrest, flow cytometry was used to analyze the cell cycle distribution in EJ-treated cells. As shown in Fig. 6, the percentage of cells in the G0/G1 phase was significantly increased from 38.74 ± 2.34% (control groups) to 53.34 ± 4.12% (cells treated with 20 μM EJ) in PC-3 cells, and from 31.59 ± 2.47% (control groups) to 48.50 ± 3.67% (cells treated with 20 μM EJ) in DU-145 cells, indicating the induction of cell cycle arrest at G0/G1 phase in prostate cancer cells.

Fig. 6

EJ arrests cell cycle at G0/G1 phase in prostate cancer cells. Cells were treated with EJ for 24 hr. After staining with PI, cell cycle distribution was analyzed through flow cytometry. Quantified histograms display the effects of EJ on cell cycle distribution. *P < 0.05, **P < 0.01, ***P < 0.001.

EJ induced caspase activation in prostate cancer cells

The MMP dissipation is usually followed by the cytochrome c release, which can activate caspase-9 and caspase-3. Then the caspase-3 effector induces the cleavage of PARP to execute the apoptotic process (Lian et al., 2018). Therefore, we next investigated the effects of EJ on activation of caspases in prostate cancer cells by western blotting analysis. As shown in Fig. 7A, the expression levels of cleaved caspase-3 and -9 were significantly up-regulated. These data suggested that EJ induced cell apoptosis through a caspase-dependent pathway.

Fig. 7

EJ induces mitochondrial caspases activation and DNA damage response in prostate cancer cells. (A) Effects of EJ on the expression of caspases in prostate cancer cells. (B) Effects of EJ on DNA damage signaling pathway in prostate cancer cells. Cancer cells were treated with various doses of EJ for 24 hr. Then total proteins were extracted for western blotting assay. Results are representative of three independent experiments.

EJ induced DNA damage responses in prostate cancer cells

DNA damage response is well-known as one of the molecular events leading to cell apoptosis. Here, we investigated the effects of EJ on DNA damage pathways in prostate cancer cells. Western blotting analysis was employed to determine the expression level of related proteins. Our results demonstrated that the expression levels of γH2AX, p-Chk1 and p-Chk2 were notably up-regulated (Fig. 7B). These data suggested that EJ induced DNA damage responses in prostate cancer cells.

DISCUSSION

Prostate cancer is one of the most commonly diagnosed and lethal malignancies in males. In the present study, we demonstrated that the compound EJ showed significant anti-cancer activity in human prostate cancer cells. The inhibitory effects of EJ on cell growth were via induction of cell apoptosis, cell cycle arrest, MMP disruption and DNA damage.

Programmed cell death via apoptosis is a fundamental cellular program that plays a critical role in physiological and pathophysiological processes. In many human cancers, apoptosis is typically disturbed, which results in an unrestricted cell proliferation. Therefore, therapeutics through induction of cancer apoptosis are a promising option for the development of novel anti-cancer agents (Fulda, 2015b). Currently, many anti-cancer agents exert their anti-tumor activities via apoptosis induction. Tivantinib induces G2/M arrest and apoptosis by disrupting tubulin polymerization in hepatocellular carcinoma (Xiang et al., 2015). Rapamycin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and induction of apoptosis (Shafer et al., 2010). Cisplatin-induced apoptosis involves membrane fluidification via inhibition of NHE1 in human colon cancer cells (Rebillard et al., 2007). Consistently, in the present study, we identified that EJ induced significant cell apoptosis in prostate cancer cells. DAPI staining demonstrated the condensed and fragmented chromatin, nuclear body fragments and irregular edges in the nuclei, indicating the induction of apoptosis in prostate cancer cells (Fig. 3). Furthermore, flow cytometric analysis demonstrated that the number of apoptotic cells was obviously increased in EJ-treated prostate cancer cells (Fig. 4). These data suggested that EJ suppressed the growth of prostate cancer cells via apoptosis induction.

As is known to all, there are two principal apoptosis signal transduction pathways: the death receptor (extrinsic) and the mitochondrial (intrinsic) pathway of apoptosis (Fulda, 2015a). In the intrinsic pathway, cytochrome c promotes activation of caspases by forming a protein complex composed of cytochrome c, Apaf-1 and caspase-9, leading to caspase-9 and subsequently caspase-3 activation (Fulda and Vucic, 2012). Then, the apoptosis process takes place. Moreover, it is reported that apoptosis induction through mitochondrial pathways is usually accompanied with the loss of MMP (Yan et al., 2017). To the best of our knowledge, the MMP is an early event preceding caspase activation, and is regarded as a hallmark of apoptosis. Induction of apoptosis via mitochondrial pathways results in the loss of mitochondrial membrane potential. Interestingly, the disruption of MMP in prostate cancer cells was detected after EJ treatment in our study (Fig. 5). Meanwhile, the activation of caspase-3 and caspase-9 was visibly observed (Fig. 7A). These results indicate that EJ induces apoptosis in prostate cancer cells via the mitochondrial pathway.

The DNA damage response (DDR) is one of the molecular events leading to cell apoptosis. Numerous anti-cancer agents have been demonstrated to induce DNA double-strand breaks in cancer cells to promote the cell apoptosis (Barbosa et al., 2018; Seah et al., 2018). In response to DNA damage, the ATM will be activated and phosphorylate the downstream substrates such as histone H2AX, cell cycle checkpoint kinases Chk-1 and Chk-2. These proteins play critical roles in regulation of cell cycle checkpoints, damaged DNA repair and activation of apoptotic pathways (Li et al., 2019). Finally, it leads to cell cycle arrest or apoptosis in the cancer cells. Importantly, induction of γH2AX has been considered as a hallmark of DNA damage response (Kudoh et al., 2010; Rudolf et al., 2013). Based on the above notion, our results indicated that the expression levels of γH2AX, p-Chk1 and p-Chk2 were significantly up-regulated, suggesting the induction of DNA damage responses in EJ-treated prostate cancer cells (Fig. 7B). Meanwhile, we also found that the cell cycle in EJ-treated prostate cancer cells was arrested at G0/G1 phase (Fig. 6). The above results suggest that EJ induces DNA damage responses in prostate cancer cells.

In summary, our study demonstrated the inhibitory effects of EJ on human prostate cancer cells. This is reflected by induction of apoptosis, cell cycle arrest, MMP disruption and DNA damage. The possible mechanism is that EJ-induced DNA damage responses in prostate cancer cells result in cell cycle arrest, MMP disruption, and apoptosis induction. However, further study is still needed to explore the exact mechanisms. The results of this study suggest that EJ is a promising agent for prostate cancer therapy in future.

ACKNOWLEDGMENTS

This study was financially supported by the Zhejiang Provincial Natural Science Foundation of China (no. LY17H310007 and Y17H280004 and LZ15H310001), Natural Science Foundation of China (no. 81774003 and 81773868), the first level (Huajun Zhao) in Zhejiang Province “151 talents project” and Qianjiang Scholar Program funded by Zhejiang Province (Huajun Zhao).

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2020 The Japanese Society of Toxicology
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