Bishonokiol A Induces Multiple Cell Death in Human Breast Cancer MCF-7 Cells

Objective: A dimeric neolignan, bishonokiol A (BHNKA) isolated from Magnolia grandiflora, significantly inhibits the proliferation of human breast cancer cells. However, the exact mechanism of BHNKA induced breast cancer cell death is unknown. In this study, we investigated the pharmacological mechanism underlying BHNKA induced MCF-7 cell death. Methods: Cell viability measurement was performed by the MTT assay. Flow cytometry with PI staining, DAPI staining, and electron microscopy were used to analyze cellular death modes. In addition, western blotting, siRNA transfection, ATP assay, and fluorescence microscopy were used to determine the mechanism of BHNKA induced MCF-7 cell death. Results: BHNKA induced cell death by apoptosis, necroptosis and autophagy at the same concentration and time in MCF-7 cells, and electron microscopy confirmed these results. The mechanism of BHNKA triggered apoptosis and autophagy in MCF-7 cells was primarily due to an increase in the Bax/Bcl-2 ratio and simultaneous up-regulation of LC3-II protein expression, respectively. BHNKA induced necroptosis by activation of the RIP1-RIP3-MLKL necroptosis cascade, up-regulation of cyclophilin D (CypD) protein expression to stimulate ROS generation. We further demonstrated that siRNA-mediated down-regulation of CypD protected against BHNKA induced cell death. Conclusions: These results suggest that BHNKA may be a potential lead compound for development as an anti-breast cancer agent for induction of multiple cell death pathways.


Introduction
Breast cancer is the most common cancer in women, threatening their health and life (Miller et al., 2019). Although chemotherapy shows positive clinical effects in breast cancer, apoptotic drug resistance, substantial sideeffects, and dose-limiting toxicity eventually reduce the chemotherapeutic efficacy (Ateba et al., 2018;Holohan et al., 2013;Kotecha et al., 2016). Therefore, research and development effort for developing highly effective and low toxicity anti-breast cancer drugs have become indispensable for saving lives.
Programmed cell death (PCD), defined as regulated cell death mediated by an intracellular program, plays crucial roles in anti-cancer therapy (Su et al., 2015). Apoptosis and autophagy are considered two types of PCD and serve as important targets for cancer chemotherapy (Levy et al., 2017;Mohamed et al., 2017). Cancer cells are initially sensitive to apoptotic induction, but develop resistance eventually (Pommier et al., 2004). Regulated necrosis, termed as necroptosis, is emerging as a new paradigm of cell death that can be activated when the
Natural products have many advantages, such as structural diversity, unique biological activities, multiple targets, low toxicity, and the ability to circumvent drug-resistance (Wang et al., 2015). Thus, the search for new anti-cancer agents from natural sources has gained importance in recent years. Bishonokiol A (BHNKA) is a dimeric neolignan isolated from Magnolia grandiflora seeds, and exhibits anti-proliferative activity, induces apoptosis, and inhibits invasion and migration of cancer cells ( Figure 1A) (Li et al., 2015;Li et al., 2019). However, the pharmacological mechanism of BHNKA induced breast cancer cell death is still unclear. In this study, we report the effect of BHNKA on MCF-7 cells and its mechanism underlying breast cancer cell death.

Cell line and cell culture
Human breast cancer MCF-7 cell line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The cells were maintained in DMEM containing 15% FBS and 1% penicillin/ streptomycin (Gibco, USA), in an atmosphere containing 5% CO 2 at 37°C.

MTT assay
The cell viability was determined by using MTT assay, as described previously (Li et al. 2019). The MCF-7 cells were seeded in 96-well plate at a density of 6,000 cells per well and treatment with various concentrations of BHNKA for 24, 48, and 72 h. At the end of each time point, MTT solution was added to each well and incubated at temperature without light. After 4 h, the MTT solution was removed, and formazan was dissolved in 100 μl of DMSO for 10 min. The absorbance (A) was determined using a microplate spectrophotometer at 570 nm (Bio-Rad, USA).

Flow cytometry
Cells were seeded at a density of 1.2×10 5 cells/well in 12-well plates. After the cells adhered, they were treated with various concentrations of BHNKA and cultured for 24 h. The cells were collected, washed with PBS, fixed with cold 75% ethanol, and stained with PI and evaluated using Accuri C6 flow cytometry (BD Biosciences, USA).

DAPI staining
Cells were seeded in 6-well plates at a density of 2×10 5 cells/well. Following treatment with various concentrations of BHNKA for 24 h, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at 4°C. Later, the cells were washed with ice-cold PBS and incubated with 500 μl of DAPI solution in the dark for 5 min. After staining, the cells were washed three times with PBS and observed using a fluorescence microscope (Olympus, Tokyo, Japan).

Electron microscopy
Cells in a 10-cm culture dish were incubated for 6 h with vehicle control or BHNKA (40 μM). After incubation, the cells were washed and fixed with 2.5% glutaraldehyde, followed by storage at 4°C. The post-fixed specimens were sent to Servicebio (China) for subsequent processing and observation by transmission electron microscopy (TEM). The processed samples were imaged for observing the submicroscopic cellular structures, as described previously (Wu et al., 2019).

Western blotting
The cells were homogenized in RIPA lysis buffer for 30 min on ice, and cell lysates centrifuged at 12,000 rpm for 30 min at 4°C. The protein concentrations measured using the BCA protein assay kit and the proteins separated on SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skimmed milk, the membranes were incubated with the primary antibodies (Bax, Bcl-2, RIP1, RIP3, MLKL, CypD, and LC3) overnight at 4 o C followed by the appropriate secondary antibodies. GAPDH was used as a loading control. An enhanced chemiluminescence kit and gel imaging equipment (Bio-Rad, USA) were used to detect the protein expression.

Detection of Mitochondrial membrane potential
MCF-7 cells were seeded in 6-well plates at a density of 2×10 5 cells/well, and then cultured with various concentrations (10, 20, and 40 μM) of BHNKA for 24 h at 37°C. After 24 h, JC-1 staining solution and culture medium were added in each well, followed by the well mixing and incubation for 20 min. After staining with JC-1, the supernatant was aspirated from the cells, followed by washes with cold JC-1 staining buffer. Following the manufacturer's protocol, the cells were imaged by fluorescence microscopy.

BHNKA exhibits anti-proliferative activity against MCF-7 cells
First, we analyzed the anti-proliferative activity of BHNKA on human breast cancer MCF-7 cells by treatment with various concentrations (0~40 μM) of BHNKA for 24, 48, and 72 h. BHNKA showed significantly inhibits the proliferation of MCF-7 cells in a concentration-and time-dependent manner, consistent with the previous study ( Figure 1) (Li et al., 2019).

BHNKA induces apoptosis in MCF-7 cells
The cell death in MCF-7 cells exposed to BHNKA for 24 h was analyzed using flow cytometry with PI staining. As shown in Figure 2A, the cell death rates were 8.0, 25.0, and 55.2% in cells treated with 20, 30, and 40 μM of BHNKA, respectively. DAPI staining of cells exposed to BHNKA showed specific morphological characteristics of apoptosis such as bright nuclear condensation ( Figure  2B). To further confirm the mode of cell death induced by BHNKA treatment, we observed the submicroscopic structure of cells under an electron microscope. As shown in Figure 2C, the cells treated with BHNKA exhibited typical characteristics of apoptosis, such as chromatin concentration and nuclear pyknosis in massive cells. Concurrently, with increasing concentrations of BHNKA, slightly greater levels of caleaved caspase-9, proapoptotic protein Bax level gradually increased, and anti-apoptotic protein Bcl-2 decreased ( Figure 2D). Furthermore, cell viability after treatment with BHNKA alone was 34.1%, whereas it increased to 60.9% after the combined treatment with BHNKA and the apoptosis inhibitor, z-VAD-fmk ( Figure 2E). These results indicated that BHNKA induced apoptosis in MCF-7 cells.

Measurement of ATP levels
MCF-7 cells were seeded into 12-well plates at a density of 2×10 5 cells/well, followed by treatment with various concentrations (10, 20, and 40 μM) of BHNKA. After 5 h, the cells were washed, lysed with RIPA lysis buffer on ice, and cell lysates centrifuged at 15,000 rpm for 5 min. The supernatant was harvested for subsequent examination. Following the ATP assay kit' protocol, ATP test solution and sample were then added to 96-well plates accompanied by well mixing, and ATP levels were determined using a luminoskan luminometer (Thermo Scientific, USA).

Measurement of ROS production
The redox-sensitive dye DCFH-DA was used to evaluate the levels of intracellular ROS, as described previously (Wu et al., 2019). MCF-7 cells were seeded in 12-well plates at a density of 2×10 5 cells/well, followed by treatment with varying concentrations (10, 20, and 40 μM) of BHNKA for 3 h. After incubation, the supernatant was removed, followed by the addition of 1 ml of diluted DCFH-DA (diluted with the serum-free medium to a final concentration of 10 μM) and incubated for 20 min at 37°C. Fluorescence intensity of the oxidized substrate was analyzed by a fluorescence microscope.

Statistical analysis
SPSS v.16.0 software (SPSS Inc., Chicago, IL, USA) was used for data analysis, and t-test was conducted to compare the data between the two test groups. P*<0.05 and P**<0.01 were considered statistically significant.

BHNKA induces necroptosis in MCF-7 cells
Interestingly, we also noticed that BHNKA induced necroptosis at the same concentration and time in MCF-7 cells. The cells treated with BHNKA (40 μM) for 24 h showed morphological characteristics of necrosis as observed by an electron microscopy such as loss of plasma membrane integrity and organelle swelling ( Figure 3A). Meanwhile, cell viability after treatment  with BHNKA alone was 33.2%, which increased to 56.6% and 58.5% after the combination treatment with BHNKA and the Nec-1 (necroptosis inhibitor) and CsA (CypD inhibitor), respectively ( Figure 3B). Furthermore, western blotting showed a significant increase in the necroptosis-related proteins (RIP1, RIP3, MLKL, and CypD) upon BHNKA treatment ( Figure 3C).

Effect of CypD on necroptosis of MCF-7 cells
Cyclophilin D (CypD) plays a critical role in the regulation of mitochondrial permeability transition and cell death (Baines et al., 2005). The CypD expression  was down-regulated significantly in MCF-7 cells after treatment with BHNKA and CsA, compared to treatment with BHNKA alone (Figure 4A and 4B). To explore the role of CypD in cell death, we investigated the effect of CypD knock-down in MCF-7 cells by siRNA. The siRNA1139 significantly silenced CypD expression leading to loss of CsA protective ability against BHNKA ( Figure 4C-4E). Altogether, these results demonstrated that BHNKA induced necroptosis in MCF-7 cells could be mimicked by down-regulation of CypD.

BHNKA induces autophagy in MCF-7 cells
BHNKA treated cells exhibited autophagic vacuoles in the cytoplasm, suggesting induction of autophagy in MCF-7 cells, while treatment with the autophagy inhibitor CQ abolished the BHNKA cytotoxicity to MCF-7 cells, as evidenced by the up-regulation of LC3-II protein expression ( Figure 5). These data indicated that BHNKA induces autophagy in MCF-7 cells.

Effect of BHNKA on mitochondrial function in MCF-7 cells
To explore whether BHNKA induced cell death is associated with mitochondrial function, we first measured the mitochondrial membrane potential (MMP), as described "Materials and Methods" section. MCF-7 cells were treated with increasing concentrations of BHNKA, and a shift in JC-1 fluorescence from red to green observed ( Figure 6A). As shown in Figure 6B, cellular ATP levels decreased to 73.4%% and 52.3%, when treated with 20 and 40 μM of BHNKA, respectively. Measurement of intracellular ROS levels using DCFH-DA showed increased fluorescence intensity of oxidized DCF after treatment with increasing concentrations of BHNKA ( Figure 6C). These results indicated that BHNKA triggered MCF-7 cell death is associated with the mitochondrial function.

Discussion
Several PCD pathways contribute to cell death, and apoptosis, the classical pathway, has emerged as an important therapeutic target for cancer cells. In cancer chemotherapy, evasion of cell death poses a significant challenge and is frequently associated with failure of cancer cells to undergo apoptosis (Hanahan et al., 2011). Cancer cells are destroyed via various death pathways; however, the mechanism of each pathway differs from one another. Moreover, cancer cells develop pathways to evade apoptosis that are specific to cancer cells only (Hu et al., 2007). For the first time, our results in MCF-7 cells show that BHNKA induced multiple cell death pathways such as apoptosis, necroptosis, and autophagy.
Autophagy is a major intracellular pathway for the degradation and recycling of cytoplasmic contents, damaged organelles, and invasive pathogens, which plays a pivotal role in the pathogenesis of many diseases, such as cancer, neuropathies, and metabolic disorders (Levine et al., 2008). Although it is essential for normal cell function and survival, tumor cells exploit autophagy for treatment resistance. However, autophagy serves as a critical pathway and demonstrates a dual role in the hallmarks of cancer (Singh et al., 2018). Combining standard chemotherapy with an autophagy inhibitor could potentially accelerate cancer cell death, in contrast, activation of autophagy enhances cancer chemotherapy sensitivity and drug-resistance (Mathiasen et al., 2002;Sun et al., 2019). In particular, our results demonstrate that BHNKA induces apoptosis and necroptosis as well as autophagy, and the induced autophagy process is related to an increased expression of LC3-II/I ratio.
Mitochondria are important organelles for cellular energy and ROS production, and play crucial roles in cell death. As an important effector molecule for apoptosis and necroptosis, CypD is regarded as a key factor for regulating the mitochondrial permeability transition pore function and cell death (Baines et al., 2005;Li et al., 2007;Porter et al., 2018). Consistently, BHNKA-induced necrotic cell death in MCF-7 cells featured a significant up-regulation of CypD expression. Down-regulation of CypD by siRNA significantly protected against BHNKA-induced cell death. Our research indicates that BHNKA-triggered PCD is closely associated with changes in mitochondrial function, mainly a decline in the mitochondrial membrane potential, decreased ATP levels, and enhanced ROS generation.
In summary, the current research showed that BHNKA triggered PCD in MCF-7 cells via induction of apoptosis, autophagy, and necroptosis. In particular, BHNKA induced necroptosis by up-regulating RIP1-RIP3-MLKL signaling pathway and CypD expression to stimulate ROS generation. Thus, BHNKA, a potential anti-breast cancer agent, could be used for future clinical applications.