Anti-proliferation of triple-negative breast cancer cells with physagulide P: ROS/JNK signaling pathway induces apoptosis and autophagic cell death

Physagulide P (PP), a new natural compound, was isolated from Physalis angulate L. in our laboratory. In this study, we demonstrated that PP potently suppressed cell proliferation by inducing G2/M phase arrest in MDA-MB-231 and MDA-MB-468 cells. Moreover, PP provoked apoptosis by decreasing the mitochondrial membrane potential and elevating the Bax/Bcl-2 protein expression ratio. The caspase inhibitor Z-VAD-FMK partly restore cell viability, suggesting that apoptosis plays as an important role in the anti-proliferative effect of PP. PP-treated cells also underwent autophagy, as evidenced by the formation of autophagosomes and the accumulation of LC3BII. Furthermore, the knockdown of LC3B reduced PP-induced cytotoxicity, indicating that autophagy played an anticancer effect. PP also induced the generation of reactive oxygen species (ROS) and resulted in c-Jun N-terminal kinases (JNK) activation. Accordingly, JNK siRNA significantly attenuated PP-triggered apoptosis and autophagy, and ROS scavengers almost completely reverse this apoptosis and autophagy. The ROS scavenger also blocked PP-induced G2/M phase arrest and the phosphorylation of JNK. Our results revealed that PP induced G2/M phase arrest, apoptosis and autophagy via the ROS/JNK signaling pathway in MDA-MB-231 and MDA-MB-468 cells. Therefore, PP is a promising candidate for the development of antitumor drugs for the treatment of triple-negative breast cancer.


INTRODUCTION
Breast cancer is the most common cancer among women worldwide [1]. In the United States, 231,840 new cases of invasive breast cancer and 40,290 deaths from this disease are estimated to occur in 2015, and one in eight women will develop breast cancer during their lifetime [2]. Triple-negative breast cancer, defined by the lack of expression of estrogen receptorα (ERα) and progesterone receptor (PR) as well as the absence of overexpression and/or gene amplification of HER2, is a unique subtype of breast cancer with limited treatment options and poor prognosis [3,4]. Compared with any other subtypes of breast cancer, triple-negative breast cancer follows a more aggressive clinical course and shows a higher risk of recurrence and death in the first 3 to 5 years after diagnosis [5]. In addition, current treatment regimens lead to chemoresistance and toxicity [6]. Hence, the development of novel therapeutic strategies as well as effective and less toxic drugs are essential in fighting triple-negative breast cancer.
Apoptosis, or type-I programmed cell death (PCD), involves the activation of catabolic enzymes -in particular proteases -in signaling cascades, which leads to cell membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies [13][14][15]. Autophagy, which is classified as the type-II PCD, is a catabolic process that sequesters cytoplasmic proteins and organelles into autophagosomes and transports them to lysosomes for recycling and degradation [16,17]. However, the ability of PP to induce apoptosis or autophagy as well as their roles and interplay in the PP-induced cell growth inhibition of triple-negative breast cancer cells remain to be determined.
ROS are mainly formed in mitochondria and are involved in the regulation of different physiological processes, including apoptosis and autophagy [18,19]. ROS can affect various signaling pathways such as mitogenactivated protein kinase (MAPK) signal transduction cascades [20,21]. Moreover, JNK, a stress-activated protein kinase (SAPK) of the MAPK family, plays a pivotal role in many cellular events, including apoptosis and autophagy [22,23]. In short, targeting the ROS/JNK signaling pathway may be effective for the treatment of triple-negative breast cancer.
Physalis angulata L., a traditional Chinese herbal medicine, is an annual herb distributed in many countries located in tropical and subtropical regions of the world and is used worldwide for its fruits. Extracts or infusions from this plant have been used in various countries in popular medicine as a treatment for a variety of illnesses, such as malaria, asthma, hepatitis, dermatitis, rheumatism, wound healing, sleeping sickness, earache, and fever, among others [24][25][26][27][28]. In the present study, we report the novel anticancer effect of PP ( Figure 1A) purified from Physalis angulata L. in triple-negative breast cancer. We further explored the molecular mechanisms, that is, the induction of G2/M phase arrest, apoptosis and autophagy mediated by the ROS/JNK signaling pathway. Moreover, a colony formation assay showed that fewer colonies formed after PP treatment, and this effect depended on dose ( Figure 1C). MDA-MB-231 and MDA-MB-468 cells were also labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) to monitor the effect of PP on cell division. After entering the cell, CFDA-SE is equally distributed between the daughter cells when the cell divides; thus, its fluorescence intensity decreases as the cell divides. As shown in Figure 1D, the CFDA-SE fluorescence intensity increased in a dose-dependent manner after treatment with PP, suggesting that PP inhibited cell division and proliferation. To further confirm the effect of PP on cell proliferation, a 5-ethynyl-20-deoxyuridine (EdU) incorporation assay was employed, Figure 1E showes that PP significantly and dose-dependently reduced the number of EdU-positive cells compared with the control group. These results show that PP inhibits the proliferation of MDA-MB-231 and MDA-MB-468 cells in a dose-and time-dependent manner.

PP induced G2/M phase arrest in triple-negative breast cancer cells
Cell cycle arrest inhibits cell proliferation. To investigate the role of cell cycle arrest in the PPmediated inhibition of cell proliferation, we examined the distribution of cell cycle in cells treated with PP by flow cytometry after staining with PI. As shown in Figure 2A, PP led to the accumulation of cells in the G2/M phase in a dose-dependent manner. To elucidate the mechanisms underlying this effect, we measured the expression levels of cell cycle-regulated proteins. A Western blotting analysis showed that PP up-regulated the expressions of p21, and down-regulated the levels of Cyclin B1 phospho-Cdc25C, Cdc25C, phospho-Cdc2 and Cdc2 ( Figure 2B). Taken together, these data suggest that the PP may alter the expression of cell-cycle related proteins to induce G2/M phase arrest and consequently inhibit the proliferation of triple-negative breast cancer cells.

PP triggered mitochondrial apoptosis in triplenegative breast cancer cells
To examine whether the cell growth inhibition induced by PP also depends on apoptosis, PP-treated cells were stained with Annexin V-Alexa Fluor 647/ propidium iodide (PI), which showed that PP treatment induced remarkable apoptosis comparing to the control group ( Figure 3A). We then measured the mitochondrial membrane potential (ΔΨ m ) by flow cytometry and found that PP treatment decreased the ΔΨ m in a dose-dependent manner ( Figure 3B). In addition to this change in ΔΨ m , increased Bax and decreased Bcl-2 protein expression were also observed after treatment with PP ( Figure 3C). ΔΨ m is well known to play an important role in the release of Cytochrome c (Cyt c). Thus, Cyt c expression was www.impactjournals.com/oncotarget further investigated by immunofluorescence. As shown in Figure 3D, Cyt c localizes to the inner mitochondrial membrane of untreated cells, but it was released into the cytosol after treatment with PP for 24 h. These results demonstrated that PP triggered apoptosis by inducing mitochondrial membrane depolarization and Cyt c release.
In the absence of functional mitochondria, apoptosomes form and activate the apoptosis process. In our study, we observed significant caspase-9, caspase-7, caspase-3 and PARP processing in total cell lysates from PP-treated cells, and the cleavage of caspase-9, caspase-7, caspase-3 and PARP markedly increased in both a time-and dose-dependent manner in MDA-MB-231 and MDA-MB-468 cells ( Figure 4A). Furthermore, when MDA-MB-231 and MDA-MB-468 cells were pre-treated with the pan-caspase inhibitor Z-VAD-FMK (10 μM) for 1 h before treatment with PP, cell viability was partly restored ( Figure 4B). Taken together, these data suggest that the cytotoxic effects of PP on MDA-MB-231 and MDA-MB-468 cells were partly caused by the activation of caspase-dependent apoptosis.

PP induced autophagic cell growth inhibition in triple-negative breast cancer cells
Autophagy, i.e., type-II PCD, plays a key role in the antiproliferative effects of external factors. As shown in   Figure 5A, the concentration-dependent formation of acidic vesicular organelles (AVOs) was evident in PP-treated cells stained with the lysosomo-tropic agent acridine orange. In addition, LC3BII expression was significantly increased and p62/SQSTM1 expression was significantly attenuated in a concentration-and time-dependent manner in both PPtreated cell lines ( Figure 5B). Consistent with the results of the Western blotting analysis, the PP-treated MDA-MB-231 and MDA-MB-468 cells exhibited an increase in the punctate pattern of LC3B and lysosome fluorescence, which represent the recruitment of LC3B into autophagosomes and the formation of autophagy lysosomes ( Figure 5C). To further demonstrate the formation of autophagy lysosomes, MDA-MB-231 and MDA-MB-468 cells were transfected with Ad-mCherry-GFP-LC3B adenovirus. Green fluorescence was almost completely quenched and appeared as red dots in PP-treated cells ( Figure 6A), indicating that PP induced the formation of autophagy lysosomes. To determine the role of autophagosome accumulation in PP-induced cytotoxicity, we knocked down LC3B, which showed that the viability of the LC3B siRNA-treated group was higher than that of the NC siRNA group ( Figure 6B). However, the apoptosis and the expression of cleaved caspase-9 and cleaved caspase-3 did not significantly differ between the LC3B siRNA group and the NC siRNA group ( Figure 6C-6D). These results suggest that autophagy acts as another way participated in triple-negative breast cancer cell growth inhibition of PP.

ROS normally exists in all aerobic cells in balance
with biochemical antioxidants, and this balance can determine the fate of cancer cells by regulating various signaling pathways, including cell cycle arrest, apoptosis, autophagy and necrosis. As shown in Figure 7A, the ROS level increased in MDA-MB-231 and MDA-MB-468 cells after treatment with different concentrations of PP for 24 h. Next, we applied NAC, a ROS inhibitor, to examine the  Figure 7C-7D). However, flow cytometry indicated that p21 siRNA failed to restore PP induced ROS generation ( Figure 7E). Furthermore, the number of apoptotic cells decreased when MDA-MB-231 and MDA-MB-468 cells were treated with NAC and PP compared with the PP-treated group ( Figure 8A). NAC also reversed the PP-induced decrease in the ΔΨ m . ( Figure  8B). Furthermore, the changes in cleaved caspase-9, cleaved caspase-7, cleaved caspase-3, cleaved PARP, Bcl-2 and Bax expression were also reversed when MDA-MB-231 and MDA-MB-468 cells were co-incubated with PP and NAC ( Figure 8C-8D). With respect to autophagy makers, LC3BII expression decreased and p62 expression increased after treatment with PP and NAC compared with the PP-treated group ( Figure 8E). Taken together, these results indicate that PP induces G2/M arrest, autophagy and apoptosis through upregulating ROS in triple-negative breast cancer cells.

PP induced JNK activation dependent on ROS production
We also investigated the effect of PP on JNK activation, and Figure 9A shows that PP increased the level of JNK phosphorylation in both MDA-MB-231 and MDA-MB-468 cells. We then used a specific JNK siRNA to assess the contribution of activated JNK to PP-induced apoptosis, autophagy or cell cycle arrest and found that the ablation of JNK with siRNA downregulated phospho-JNK in MDA-MB-231 cells, irrespective of PP treatment ( Figure 9B). In addition, the number of PP-triggered apoptotic cells decreased when cells were transfected with JNK siRNA compared to the NC siRNA group ( Figure  9C). When MDA-MB-231 cells were transfected with JNK siRNA and co-treated with PP, JNK siRNA blocked PP-induced caspase activation ( Figure 9D). Similarly, JNK siRNA also blocked the PP-induced decrease in ΔΨ m and the Bax/Bcl-2 ratio ( Figure 9E-9F). Moreover, we found that the ablation of JNK decreased the effected of PP on LC3BΙΙ ( Figure 9G). However, flow cytometry indicated that JNK siRNA failed to restore the PP-induced increase in G2/M phase arrest ( Figure 9H). These results suggest that the activation of JNK is required for PP-induced apoptosis and autophagy but not involved in G2/M phase arrest. Subsequently, the relationship between ROS generation and JNK activation in PP-induced apoptosis and autophagy was investigated by treating of MDA-MB-231 and MDA-MB-468 cells with PP for 24 h alone or in combination with NAC. Changes in phospho-JNK expression were completely reversed when triple-negative breast cancer cells were treated with NAC ( Figure 9I). In addition, JNK siRNA failed to restore PP induced ROS generation ( Figure 9J). These results reveal that ROS is the proximal event of JNK and most likely initiates PPinduced apoptosis and autophagy. Thus, PP-induced ROS generation activates JNK signaling to inhibit the growth of triple-negative breast cancer cells.

DISCUSSION
PP is a novel withanolide compound that was isolated from Physalis angulate L at our laboratory, and its analogues have been reported to be cytotoxic to many types of cancer cells via diverse mechanisms [29,30]. In this study, we investigated the effect of PP on MDA-MB-231 and MDA-MB-468 cells. Our results confirm that PP inhibits proliferation of triple-negative breast cancer cells via G2/M arrest, apoptosis and autophagy mediated by ROS/JNK signaling.
Cell cycle deregulation is a hallmark of cancer, and the induction of cell cycle arrest, especially G2/M phase arrest, may be an effective strategy to treat aberrant cancer cell proliferation [31][32][33]. The Cyclin B1 and Cdc2 complex, which is rendered inactive by the phosphorylation of Cdc2, plays a key role in promoting the G2/M phase transition [11]. At the initiation of mitosis, cell division cycle protein (Cdc25C) is activated to dephosphorylate of Cdc2 [34,35], and Cdc25C is phosphorylated by the checkpoint kinases Chk1 and Chk2 and downregulates Cdc25C activity [36,37]. In addition, p21 plays a critical role in the G2/M checkpoint by inhibiting the Cdc2/cyclinB1 complex [38]. According to our flow cytometric analysis, PP treatment resulted in G2/M phase arrest in triple-negative breast cancer cells. Further Western blotting analyses showed that PP caused G2/M phase arrest by increasing the protein levels of p21 while decreasing the protein levels of Cdc2, phospho-Cdc2, phospho-Cdc25C, Cdc25C and CyclinB1.
Apoptosis is an evolutionarily conserved cell suicide mechanism to eliminate redundant, damaged, or infected cells [39] and can be activated by compounds targeting extrinsic and intrinsic apoptosis pathways [40]. The extrinsic apoptotic pathway involves the interaction of ligands with their respective cell surface death receptors, such as Fas/CD95 and TNFR1, which results in the activation of the proteases caspase-9, caspase-3 and other downstream caspases and the initiation of the apoptotic cascade [41]. Alternatively, the mitochondriamediated intrinsic pathway may trigger apoptosis. Our results showed that ΔΨ m sharply decreased after PP treatment, indicating that PP depolarized mitochondria in MDA-MB-231 and MDA-MB-468 cells. Mitochondrial membrane depolarization can release Cyt c from the mitochondrial intramembrane space to the cytosol and activate cytosolic caspases [42][43][44].
Autophagy, another caspase-independent antiproliferation pathway, plays a significant role in determining www.impactjournals.com/oncotarget cellular fate [45]. We found that PP induced autophagy, as evidenced by the accumulation of AO-staining acidic vesicles, the formation of autophagy lysosomes observed with an imageXpress® confocal microscope and the upregulation of LC3BII. To further explore the relationship between autophagy and cell growth inhibition, we transfected MDA-MB-231 cells with LC3B siRNA, which showed that LC3B knockdown markedly attenuated PP-mediated decreases in cell vitality, indicating that autophagy plays an anti-proliferative role. However, the LC3B siRNA-mediated decrease in autophagy did not affect the number of apoptotic cells and protein levels of cleaved caspase-9 and cleaved caspase-3, which indicates that autophagy and apoptosis independently inhibit cell growth.  The generation of ROS in MDA-MB-231 and MDA-MB-468 cells is a striking feature of PP-induced apoptosis and autophagy. Specifically, ROS are important signaling molecules that regulate many signal-transduction pathways [46][47][48], and excessive ROS generation may interfere with cellular signaling pathways and play critical roles in inducing both cell apoptosis and autophagy [49,50]. Furthermore, emerging evidences illustrated that natural compounds such as Physalin A [51], withaferin A [27] and celastrol [11] can cause ROS-mediated apoptosis and autophagy due to their structure of α, β-unsaturated ketone moieties [52]. PP also has an α, β-unsaturated ketone moiety, thus we hypothesized that PP-induced apoptosis and autophagy may be mediated by ROS. In this study, PP significantly increased ROS generation in MDA-MB-231 and MDA-MB-468 cells, and the ROS inhibitor NAC reversed the PP-induced inhibition of cell proliferation. NAC blocked PP-induced G2/M arrest, apoptosis and autophagy in MDA-MB-231 and MDA-MB-468 cells, suggesting that PP affected cell proliferation via the ROS-mediated modulation of the cell cycle, apoptosis and autophagy.
JNK, a member of the MAPK family, transduces cellular signals in response to different physiological stimuli and stresses [49]. To explore the relationship between ROS and JNK as well as their role in G2/M phase arrest, the ability of PP to activate JNK was investigated. PP induced ROS production in MDA-MB-231 and MDA-MB-468, which strongly activated JNK (phosphorylation). Moreover, NAC almost completely reversed the PP-induced inhibition of apoptosis and autophagy, and JNK siRNA significantly attenuated these two processes. NAC, but not JNK siRNA, also strongly blocked G2/M phase arrest, indicating that ROS, but not JNK, mediated PP-induced cell cycle arrest. In addition, NAC completely blocked JNK phosphorylation, implying that ROS is the proximal event of JNK signaling. In summary, our studies provide experimental evidence to support that PP induces apoptosis and autophagy via the ROS/JNK signaling pathway and that ROS play a vital role in G2/M phase arrest caused by PP.
Our study showed that PP suppresses cell proliferation in triple-negative breast cancer cells by causing G2/M phase arrest, apoptosis and autophagy. In addition, PP induced apoptosis and autophagy by activating ROS/JNK signaling. Moreover, inhibiting autophagy with LC3B siRNA attenuated PP-induced cell growth inhibition, indicating that PP-induced autophagy is an anti-proliferative process in cells ( Figure 10). These

Cell culture and reagents
The triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231and MDA-MB-468 were cultured in DMEM and L-15 media, respectively. The media were supplemented with 10% fetal bovine serum, 50 μg/ml penicillin and 50 μg/ml streptomycin at 37 °C and 5% CO 2 in a humidified environment.

Cell viability and tumor colony forming assays
MDA-MB-231 and MDA-MB-468 cells were treated with various concentrations of PP or 0.1% DMSO. Cell viability and tumor colony formation assays were performed as previously described [49].

EdU incorporation assay
MDA-MB-231 and MDA-MB-468 cells were treated with various concentrations of PP or 0.1% DMSO. The EdU incorporation assay was performed using an EdU labeling/detection kit (Ribobio, Guangzhou, China). The operating methods were performed as previously described [53]. The cells were subsequently observed with an imageXpress ® confocal microscope (Molecular Devices, USA).

Cell cycle analysis
In brief, after treatment with different concentrations of PP, the cells were harvested and fixed with 70% ethanol at -20 °C overnight. The cells were then stained with PI/ RNase Staining Buffer (Beyotime, Shanghai, China) for 15 min and analyzed with a BD Accuri C6 flow cytometer.

Measurement of ROS
To measure the intracellular ROS levels, MDA-MB-231 and MDA-MB-468 cells were pretreated with various concentrations of PP or 0.1% DMSO for 24 h and then incubated in the dark with 10 mM of the oxidationsensitive fluorescent probe 2'7'-dichlorfluoresceindiacetate (DCFH-DA, Beyotime) for 20 min at 37 °C. DCFH-DA was cleaved by intracellular esterase to liberate free DCFH, and the ROS level was measured using a BD Accuri C6 flow cytometer.

Measurement of the ΔΨm
ΔΨ m was assessed with a JC-1 assay kit (KeyGen Biotech, Nanjing, China) as previously described [54]. Mitochondrial depolarization was measured by a decrease in the red/green fluorescence intensity ratio.

Acridine orange (AO) staining
MDA-MB-231 and MDA-MB-468 cells were seeded in a 6-well plate and incubated with various concentrations of PP for 24 h. The cells were then harvested and stained with AO for 15 min. After washing with PBS, the cells were analyzed with a BD Accuri C6 flow cytometer.

LC3B immunofluorescence
MDA-MB-231 and MDA-MB-468 cells were plated in 96-well culture plates at a density of 2 × 10 4 cells per well and treated with different concentrations of PP for 18 h. The cells were then washed with PBS containing 0.05% saponin, fixed with 4% paraformaldehyde for 30 min, incubated with 0.5% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. The cells were then incubated with antibodies against LC3B (Cell Signaling Technology, 1:50) overnight at 4 °C; the nuclei were stained with Hoechst 33258 (Yeasen Biotechnology, Shanghai, China), and lysosomes were stained with Lysotracker for 30 min before imaging. Finally, images were taken with an imageXpress ® confocal microscope.

Ad-mCherry-GFP-LC3B transfection
Cells were grown on 96-well plates until they reached 60%-70% confluence and then transfected with Ad-mCherry-GFP-LC3B adenovirus (Beyotime) at an MOI of 30 for 6 h at 37 °C. Following treatment with PP for 18 h, the cells were stained with Hoechst 33342 (Yeasen). Finally, images were taken with an imageXpress ® confocal microscope.

Western blotting analysis
MDA-MB-231 and MDA-MB-468 cells were lysed in western blotting lysis buffer and centrifuged at 12000 rpm for 10 min. Proteins were extracted for western blot as previously described [55]. Bound immuno-complexes were detected with a ChemiDOC TM (Bio-Rad Laboratories, Hercules, CA).

Cyt c immunofluorescence
MDA-MB-231 and MDA-MB-468 cells growing in glass-bottom dishes were treated with different concentrations of PP for 24 h. The cells were then fixed in 4% paraformaldehyde, washed with PBS, incubated with 0.5% Triton X-100 and blocked with 5% BSA. The cells were then incubated with primary antibodies against Cyt c (Cell Signaling Technology, 1:200) overnight at 4 °C, followed by incubation with Alexa Flour 488-conjugated secondary antibody (Yeasen) for 2 h at room temperature in the dark. The nuclei were stained with Hoechst 33258, and lysosomes were stained with Mitotracker for 30 min before imaging. A laser scanning confocal microscope LSM 700 was used to analyze co-localization (LSM700, Carl Zeiss, Germany).

Statistical analysis
All experiments were conducted more than three times. The results were analyzed using a one-way or twoway ANOVA followed by Tukey's range test. The data are presented as the mean ± standard error, and significance was set to p < 0.05.

Author contributions
PY and CZ conceived or designed the experiments, performed the experiments, wrote the manuscript, and analyzed the data. TM performed the experiments and analyzed the data. HZ performed the experiments. YY analyzed the data. CG and MZ provided the tested drugs. LY analyzed the data and wrote the manuscript. LK conceived or designed the experiments. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS
We thank the Cellular and Molecular Biology Center of China Pharmaceutical University for Assistance with confocal microscopy work and we are grateful to Xiao-Nan Ma for her technical help.

CONFLICTS OF INTEREST
The authors have no conflicts of interest.