Antiproliferation for Breast Cancer Cells by Ethyl Acetate Extract of Nepenthes thorellii x (ventricosa x maxima)

Extracts from the Nepenthes plant have anti-microorganism and anti-inflammation effects. However, the anticancer effect of the Nepenthes plant is rarely reported, especially for breast cancer cells. Here, we evaluate the antitumor effects of the ethyl acetate extract of Nepenthes thorellii x (ventricosa x maxima) (EANT) against breast cancer cells. Cell viability and flow cytometric analyses were used to analyze apoptosis, oxidative stress, and DNA damage. EANT exhibits a higher antiproliferation ability to two breast cancer cell lines (MCF7 and SKBR3) as compared to normal breast cells (M10). A mechanistic study demonstrates that EANT induces apoptosis in breast cancer cells with evidence of subG1 accumulation and annexin V increment. EANT also induces glutathione (GSH) depletion, resulting in dramatic accumulations of reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX), as well as the depletion of mitochondrial membrane potential (MMP). These oxidative stresses attack DNA, respectively leading to DNA double strand breaks and oxidative DNA damage in γH2AX and 8-oxo-2′deoxyguanosine (8-oxodG) assays. Overall these findings clearly revealed that EANT induced changes were suppressed by the ROS inhibitor. In conclusion, our results have shown that the ROS-modulating natural product (EANT) has antiproliferation activity against breast cancer cells through apoptosis, oxidative stress, and DNA damage.


Introduction
Breast cancer is the most common cancer in women, comprises 30% of all new female cancer cases, and accounts for 15% of all female cancer-related deaths [1]. Breast cancer is complex and an effective cure remains elusive. Resistance to clinical drugs and radiation reduces the therapeutic effect against breast cancer and is partly attributed to the loss of apoptosis function [2]. Accordingly, apoptosis-inducing drugs may improve the therapeutic effect against breast cancer cells.
Accumulating evidence suggests that natural products contain many bioactive components for cancer prevention and therapy, and sometimes provide advantages over isolated compounds. This is partly because natural products consist of many bioactive components that can suppress the function of multiple targets [3]. Natural products provide valuable resources for drug discovery for breast cancer treatment through apoptosis [2,[4][5][6][7]. For example, Phyla nodiflora L. extracts were found to inhibit cell proliferation by inducing apoptosis in human breast cancer cells [2]. The study of additional natural products for drug discovery against breast cancer cells is thus warranted.
Nepenthes (also known as tropical pitcher plants) are tropical carnivorous plants. Nepenthes comprise a number of natural and cultivated hybrids and present diverse species development. Many species of Nepenthes are commonly used in herbal medicine in several Southeast Asian countries [8]. Some types of extracts from Nepenthes are known to have anti-bacterial and anti-fungal properties. For example, methanolic extract of N. bicalcarata inhibited growth of gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis and B. spizizenii) with the minimum inhibitory concentration (MIC) at 256 µg/mL [9]. Hexane extract of N. ventricosa x maxima inhibited the growth of several species of fungi such as Alternaria alternata, Aspergillus niger, Bipolaris oryzae, Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani, Rhizopus stolonifer var. stolonifera, and Sclerotinia sclerotiorum with MIC values ranging from 7.2 to 43.7 µg/mL [10]. Nepenthes extracts have been reported to suppress inflammation [11]. Anti-inflammation drugs frequently show anticancer effects [12][13][14]. Recently, the methanol extract and its sequential partitions of N. alata Blanco as well as its bioactive compound plumbagin demonstrated the anti-breast-cancer effect [15]. Therefore, we hypothesize that extracts from other Nepenthes hybrid may have an anticancer effect against breast cancer cells.
This study evaluates the antiproliferation effect from an ethyl acetate extract of Nepenthes thorellii x (ventricosa x maxima) (EANT) on breast cancer cells. The underlying mechanisms of antiproliferation (e.g., cell viability, apoptosis, oxidative stress, and DNA damage) were determined on breast cancer cells following EANT treatment.

The Identified Components from Fingerprint Profiles of EANT
According to HPLC fingerprinting assay (Supplementary Figure S1), the major bioactive components of EANT are isoplumbagin, cis-isoshinanolone, quercetin 3-O-(6"-n-butyl β-D-glucuronide), and fatty acids. In the present study, we focused on the antiproliferation effect of breast cancer cells using EANT and therefore the detailed involvement of these bioactive components on anti-breast cancer effect was not addressed.

EANT Preferentially Inhibits Viability of Breast Cancer Cells
In Figure 1A, EANT dose-dependently more significantly reduces the viability (%) of two breast cancer cells (MCF7 and SKBR3) than normal breast cells (M10). In Figure 1B, the viability reducing effects of EANT are suppressed by the inhibitor pretreatments for ROS (NAC). Accordingly, the role of oxidative stress and apoptosis in EANT-treated breast cancer cells warrants further detailed investigation. For comparison to clinical drugs, the cell viability of breast cancer cells treated with cisplatin was performed ( Figure 1C). The drug sensitivity of EANT in breast cancer cells is higher than that of cisplatin at 24 h treatment. The drug sensitivity of cisplatin in breast cancer cells at 48 and 72 h is higher than that of 24 h treatment.
of oxidative stress and apoptosis in EANT-treated breast cancer cells warrants further detailed 96 investigation. For comparison to clinical drugs, the cell viability of breast cancer cells treated with 97 cisplatin was performed ( Figure 1C). The drug sensitivity of EANT in breast cancer cells is higher 98 than that of cisplatin at 24 h treatment. The drug sensitivity of cisplatin in breast cancer cells at 48 99 and 72 h is higher than that of 24 h treatment.     Figure 2A shows the flow cytometry patterns of cell cycle distribution in breast cancer cells (MCF7 and SKBR3) without (up) or with (down) NAC pretreatment. In Figure 2B, the subG1 and G2/M population gradually accumulates and the G1 population gradually decreases in breast cancer cells after EANT treatments. After NAC pretreatments, the subG1 accumulation and cell cycle disturbance recover to the normal distribution as control.

EANT Induces Apoptosis in Breast Cancer Cells
The possibility that subG1 accumulation may lead to apoptosis was further examined by flow cytometry. Figure 3A shows the flow cytometry patterns of annexin V/7AAD in breast cancer cells (MCF7 and SKBR3). In Figure 3B (top part), the early apoptosis (%) (annexin V (+)/7AAD (-)) of MCF7 cells is dramatically increased to about 80% in 15 µg/mL of EANT and its late apoptosis (%) (annexin V (+)/7AAD (+)) is increased to 20% compared to the control. In Figure 3B (bottom part), the early and late apoptosis (%) of SKBR3 cells is only mildly increased in 15 µg/mL of EANT compared to the control. In a higher concentration (25 µg/mL), EANT is more likely to induce late apoptosis than early apoptosis in both breast cancer cells. Figure 3C,E illustrate the roles of ROS and apoptosis effects on flow cytometry patterns of annexin V/7AAD in breast cancer cells (MCF7 and SKBR3). At 12 h of EANT treatment, the early apoptosis population exceeds the late apoptosis population in breast cancer cells. At 24 h of EANT treatment, the early apoptosis population shifts to late apoptosis in breast cancer cells. In Figure 3D,F, the early apoptosis population, that is, annexin V(+)/7AAD (−) (%), increases dramatically for 12 h and then declines at 24 h in breast cancer cells. The late apoptosis population, that is, annexin V(+)/7AAD (+) (%), increases dramatically for 12 and 24 h in breast cancer cells. For each cycle phase, treatments labeled without the same lower-case letters indicate significant 119 difference. p < 0.05~0.0001. Data, mean ± SD (n = 3). Positive controls for subG1 accumulation and

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The possibility that subG1 accumulation may lead to apoptosis was further examined by flow 123 cytometry. Figure 3A shows the flow cytometry patterns of annexin V/7AAD in breast cancer cells 124 (MCF7 and SKBR3). In Figure 3B (top part), the early apoptosis (%) (annexin V (+)/7AAD (-)) of 125 MCF7 cells is dramatically increased to about 80% in 15 μg/mL of EANT and its late apoptosis (%)

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(annexin V (+)/7AAD (+)) is increased to 20% compared to the control. In Figure 3B   For EANT post-treatment for 12 h, NAC or Z-VAD pretreatment inhibits both early and late apoptosis populations. For EANT post-treatment for 24 h, NAC or Z-VAD pretreatment inhibits late apoptosis populations and the late apoptosis population shifts to early apoptosis.

EANT Induces ROS Production and GSH Depletion in Breast Cancer Cells
Since NAC reverts the EANT induced changes of cell viability, cell cycle distribution, and apoptosis, the status of oxidative stress such as the ROS level in EANT-treated breast cancer cells warrants detailed investigation. Figure 4A,C respectively show flow cytometry patterns of ROS in breast cancer cells (MCF7 and SKBR3) without or with NAC pretreatment. In Figure 4B, the ROS (+) (%) is dramatically increased in both 15 and 25 µg/mL of EANT. In Figure 3D, the ROS (+) (%) increases dramatically within 5 min and is suppressed by NAC pretreatment in breast cancer cells (MCF7 and SKBR3).
Because glutathione (GSH) has a ROS-scavenging function [16], the status of GSH level in EANT-treated breast cancer cells warrants detailed investigation. Figure 4E shows flow cytometry patterns of GSH in breast cancer cells (MCF7 and SKBR3). In Figure 4F, the GSH (+) (%) is dramatically decreased in 25 µg/mL of EANT in breast cancer cells.

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Because glutathione (GSH) has a ROS-scavenging function [16], the status of GSH level in 160 EANT-treated breast cancer cells warrants detailed investigation. Figure 4E shows flow cytometry 161 patterns of GSH in breast cancer cells (MCF7 and SKBR3). In Figure 4F,

EANT Induces MitoSOX Production and MMP Reduction in Breast Cancer Cells
Since NAC reverts the EANT induced changes to cell viability, cell cycle distribution, and apoptosis, the status of oxidative stress such as MitoSOX and MMP levels in EANT-treated breast cancer cells warrants detailed investigation. Figure 5A shows flow cytometry patterns of MitoSOX in breast cancer cells (MCF7 and SKBR3). In Figure 5B, the MitoSOX (+) (%) is dramatically increased in both 15 and 25 µg/mL of EANT. Figure 5C,E respectively show flow cytometry patterns of MitoSOX in breast cancer cells (MCF7 and SKBR3) without or with NAC or MitoTEMPO. In Figure 5D,F, the MitoSOX (+) (%) of breast cancer cells are suppressed by the inhibitor pretreatments for ROS (NAC) or MitoSOX (MitoTEMPO). Figure 5G shows flow cytometry patterns of MMP in breast cancer cells (MCF7 and SKBR3). In Figure 5H, the MMP (−) (%) is dramatically increased in both 15 and 25 µg/mL of EANT. Figure 5I shows the time course for flow cytometry patterns of MMP in breast cancer cells (MCF7 and SKBR3) without or with NAC. In Figure 5J, the MMP (−) (%) of breast cancer cells is increased at 12 and 24 h and is suppressed by the NAC pretreatment.

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ROS is a DNA damage factor [17] and the DNA damage status in EANT-treated breast cancer 204 cells warrants detailed investigation. Figure 6A shows flow cytometry patterns of γH2AX in breast 205 cancer cells (MCF7 and SKBR3). In Figure 6B, the γH2AX (+) (%) is dramatically increased in both 15 206 and 25 μg/mL of EANT. Figure 6C shows time course for flow cytometry patterns of γH2AX in 207 breast cancer cells (MCF7 and SKBR3) without or with NAC pretreatment. In Figure 6D

EANT Induces DNA Damage in Breast Cancer Cells
ROS is a DNA damage factor [17] and the DNA damage status in EANT-treated breast cancer cells warrants detailed investigation. Figure 6A shows flow cytometry patterns of γH2AX in breast cancer cells (MCF7 and SKBR3). In Figure 6B, the γH2AX (+) (%) is dramatically increased in both 15 and 25 µg/mL of EANT. Figure 6C shows time course for flow cytometry patterns of γH2AX in breast cancer cells (MCF7 and SKBR3) without or with NAC pretreatment. In Figure 6D, the γH2AX (+) (%) of breast cancer cells are suppressed by NAC pretreatment. Figure 6E shows flow cytometry patterns of 8-oxodG in breast cancer cells (MCF7 and SKBR3). In Figure 6F, the 8-oxodG (+) (%) is gradually increased in both 15 and 25 µg/mL of EANT. Figure 6G shows time course for flow cytometry patterns of 8-oxodG in breast cancer cells (MCF7 and SKBR3) without or with NAC pretreatment. In Figure 6H, the 8-oxodG (+) (%) of breast cancer cells is increased at 12 and 24 h and is suppressed by NAC pretreatment.

EANT Preferentially Inhibits Proliferation of Breast Cancer Cells
The antiproliferation effect of cancer cells using Nepenthes has been rarely reported. The current study demonstrates that EANT inhibited the cell proliferation of breast cancer cells more than that of normal breast cells at higher concentrations (15 and 25 µg/mL) ( Figure 1A). IC 50 values of EANT are respectively 10 and 15 µg/mL for MCF7 and SKBR3 cells at 24 h MTS assay. For comparison to extracts from other Nepenthes such as N. alata Blanco, IC 50 values at 24 h MTT assay using MCF7 cells showed 99.78 µg/mL for its methanol extract and showed 36.60, 90.02, 61.13, and > 100 µg/mL for its CH 2 CL 2 , EtOAc, n-BuOH, and H 2 O fractions, respectively [15]. These results suggest that our developed ethyl acetate extract of N. thorellii x (ventricosa x maxima), that is, EANT, has higher sensitivity to breast cancer MCF7 cells than that of N. alata Blanco. It is possible that extracts from different Nepenthes may have different components or proportions leading to different drug sensitivity to breast cancer cells.
For comparison to clinical drugs, the IC 50 value of cisplatin is 15 µg/mL for SKBR3 cells but it is undetectable for MCF7 cells ( Figure 1C). IC 50 value of cisplatin at 48 and 72 h MTS assay is 3.71 and 2.69 µg/mL for SKBR3 cells and 10.45 and 6 µg/mL for MCF7 cells ( Figure 1C). Similarly, IC 50 values of cisplatin at 72 h treatment by MTT assay are 3.36 and 4.44 µg/mL [24] for SKBR3 and MCF7 cells. Although the IC 50 values of EANT at 24 h treatment are higher than that of cisplatin at 72 h treatment, their treatment times are different. For 24 h treatment, EANT has higher drug sensitivity to MCF7 cells and shows similar sensitivity to SKBR3 cells compared to cisplatin.

EANT Induces Oxidative Stress on Breast Cancer Cells
Oxidative stress is an imbalance status between ROS production and antioxidant response [25]. When ROS is overproduced, the cellular antioxidant system is unable to remove extra ROS to steady its state, thus reducing the antioxidant level. The current study found that intracellular ROS and mitochondrial superoxide MitoSOX were upregulated and MMP was downregulated following EANT treatment. These results suggest that EANT induces oxidative stress in breast cancer cells (MCF7 and SKBR3). These oxidative stress changes were validated by NAC or MitoTEMPO pretreatments.
GSH is an ROS scavenging antioxidant in cells [16] to maintain oxidative homeostasis. When the ROS level exceeds the ROS cleanness ability of GSH, the intracellular ROS level may increase and the GSH level may decrease. Natural products have been reported to inhibit cellular antioxidant system of cancer cells and lead to cell death for cancer treatments [26]. For example, ethanolic extract of red algae Gracilaria tenuistipitata can increase ROS production and decrease the GSH level of oral cancer cells [27]. Consistently, we found that GSH level declined dramatically at higher concentration of EANT (25 µg/mL). It is noted that the GSH level was not completely depleted in breast cancer cells (MCF7 and SKBR3) following EANT treatment. In addition to GSH, this warrants a detailed investigation for the involvement of other non-GSH members in antioxidant systems.
Plumbagin is a common naphthoquinone in Nepenthes, however, isoplumbagin but not plumbagin is identified in EANT. Plumbagin was reported to bind to GSH [28], behave as electrophile against GSH [29], and induce GSH depletion of leukemia Kasumi-1 [30] and U937 [31] cells. However, the GSH effect of isoplumbagin remains unclear. Accordingly, the role of the naphthoquinones isoplumbagin in cytotoxic effects against breast cancer cells needs detailed investigation in future. Moreover, the possible involvement of the other main compounds (cis-isoshinanolone and quercetin 3-O-(6"-n-butyl β-D-glucuronide)) in EANT-induced cytotoxicity of breast cancer cells cannot be excluded.

EANT Induces Oxidative Stress-Mediated Apoptosis and DNA Damage on Breast Cancer Cells
Oxidative stress is an activator for triggering apoptosis [32]. SubG1 accumulation is one of the apoptosis indicators [33]. In the current study, EANT partly induces subG1 accumulation and increases the intensity of annexin V. These apoptosis events were suppressed by NAC pretreatment, suggesting that EANT induces apoptosis of breast cancer cells depending on oxidative stress.
Moreover, oxidative stress is an activator for triggering DNA damage [34]. In the current study, the γH2AX marker [35] for DNA double strand breaks was induced in breast cancer cells after EANT treatment. The role of oxidative stress in DNA double strand breaks was further confirmed by evidence that NAC pretreatment suppresses the γH2AX intensity in breast cancer cells (MCF7 and SKBR3) following EANT treatment. This finding raises the possibility that oxidative stress may attach DNA to generate other types of DNA damage. In the example of 8-oxodG [36], we further demonstrated that oxidative DNA damage is also induced in breast cancer cells after EANT treatment. Again, this type of oxidative DNA damage in breast cancer cells is suppressed by NAC pretreatment. Therefore, EANT induces apoptosis and DNA damage to breast cancer cells through oxidative stress.

Conclusion
Extracts from Nepenthes plants have been reported to have anti-microorganism effects, but the anticancer effect of Nepenthes has rarely been investigated. This study examines the anticancer effect of Nepenthes on breast cancer cells in the example of ethyl acetate fraction of N. thorellii x (ventricosa x maxima) (EANT). EANT shows a greater antiproliferation effect against breast cancer cells than for normal breast cells. Different flow cytometric assays show that EANT induces oxidative stress and apoptosis in breast cancer cells and these changes were suppressed by their inhibitors. In conclusion, EANT exerts an antiproliferation effect on breast cancer cells and the detailed mechanisms as mentioned are explored in a ROS-dependent manner.

Nepenthes Extraction and Inhibitors
The aerial parts of Nepenthes thorellii x (ventricosa x maxima) were collected from Dr. Celica Koo Botanic Conservation Center (KBCC), Pingtung County, Taiwan, in October 2014. This Nepenthes hybrid plant was identified by KBCC researcher Ray-Hsuan Kuo, marked with the voucher number K47512 EA, and stored in the Graduate Institute of Natural Products, Kaohsiung Medical University.
The aerial part of plant material (500 g) was sliced and immersed in MeOH (1 L) for three days. The organic solvent was removed by a rotavapor (Heidolph, Schwabach, Germany) to afford a methanol extract (25 g). This extract was partitioned between water and ethyl acetate (EA). The EA-soluble fraction of N. thorellii x (ventricosa x maxima) (9.5 g) is referred to here as EANT. EANT was stored in −20 • C and dissolved in dimethyl sulfoxide (DMSO) prior to experimentation. All treatments have the same concentration of DMSO, that is, 0.05%.
To examine the role of oxidative stress, cells were pretreated with 2 mM ROS inhibitor N-acetylcysteine (NAC) (Sigma-Aldrich; St Louis, MO, USA) [37] for 1 h. The role of mitochondrial oxidative stress was also examined by pretreating cells with 10 µM of the mitochondrial superoxide inhibitor MitoTEMPO (Cayman Chemical, Ann Arbor, Michigan, USA) [38] for 1 h. The role of apoptosis was examined by pretreating cells with 20 µM of an apoptosis inhibitor Z-VAD-FMK (Z-VAD) (Selleckchem. com; Houston, TX, USA) [39] for 2 h.

HPLC Fingerprint Profile of EANT
In order to understand the abundance and distribution of the components of EANT, the fingerprint profile of EANT was studied. A Shimadzu LC-20AD prominence liquid chromatograph HPLC equipped with SPD-M10A VP diode array detector was used for investigation. Injection of EANT (volume = 100 µL, concentration = 1 mg/mL, in MeOH) was performed by a Shimadzu SIL-10AD VP auto injector. The separation of EANT was executed by a Phenomenex Luna 5 µ C18(2) 100 Å column  Figure S1 and Table S1, respectively.
The plant material of N. thorellii x (ventricosa x maxima) was collected from KBCC. However, it is hard to obtain large amounts of this plant for active component separation and identification. Therefore, we bought another Nepenthes plant (N. mirabilis) from a local drug store in China. Because taxonomically related plants usually produce similar secondary metabolites (chemotaxonomic approaches), we used the major compounds isolated from N. mirabilis as standard to interpret the components in the fingerprint profile of our research material. We also used repeated column chromatography to isolate those major compounds from N. mirabilis. Those major compounds were identified by their NMR and mass spectroscopic data.

Cell Culture and Viability
Human breast cancer cells (MCF7 and SKBR3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY, USA). Human breast normal cells (M10) were obtained from Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan) and cultured in Minimum Essential Medium Alpha Medium (αMEM) (Gibco). All cells were supplemented with 10% fetal bovine serum (Gibco) and antibiotics, and maintained in a humidified 5% CO2 incubator at 37 • C. Cell viability was determined by Promega MTS Assay (Madison, WI, USA) as previously described [27].