Nepenthes Ethyl Acetate Extract Provides Oxidative Stress-Dependent Anti-Leukemia Effects

Several kinds of solvents have been applied to Nepenthes extractions exhibiting antioxidant and anticancer effects. However, they were rarely investigated for Nepenthes ethyl acetate extract (EANT), especially leukemia cells. The purpose of the present study was to evaluate the antioxidant properties and explore the antiproliferation impact and mechanism of EANT in leukemia cells. Five standard assays demonstrated that EANT exhibits antioxidant capability. In the cell line model, EANT dose-responsively inhibited cell viabilities of three leukemia cell lines (HL-60, K-562, and MOLT-4) based on 24 h MTS assays, which were reverted by pretreating oxidative stress and apoptosis inhibitors (N-acetylcysteine and Z-VAD-FMK). Due to similar sensitivities among the three cell lines, leukemia HL-60 cells were chosen for exploring antiproliferation mechanisms. EANT caused subG1 and G1 cumulations, triggered annexin V-detected apoptosis, activated apoptotic caspase 3/7 activity, and induced poly ADP-ribose polymerase expression. Moreover, reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane depolarization were generated by EANT, which was reverted by N-acetylcysteine. The antioxidant response to oxidative stress showed that EANT upregulated mRNA expressions for nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), thioredoxin (TXN), heme oxygenase 1 (HMOX1), and NAD(P)H quinone dehydrogenase 1 (NQO1) genes. Moreover, these oxidative stresses led to DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) and were alleviated by N-acetylcysteine. Taken together, EANT demonstrated oxidative stress-dependent anti-leukemia ability to HL-60 cells associated with apoptosis and DNA damage.


Introduction
Leukemia is a type of cancer that generates abnormal, immature blood cells derived from bone marrow dysfunction and carcinogenesis. Leukemia is classified into acute and chronic types with different cell differentiation [1]. The acute types contain acute myeloid leukemia (AML) [2] and acute lymphoblastic leukemia (ALL) [3]. AML is common in adults, and ALL is common in children [4]. Cancer Statistics from 2019 show that leukemia in males and females rank ninth and tenth, respectively, for estimated new cases, and high mortality ranks sixth and eighth, respectively, for estimated deaths [5]. Leukemia therapy includes the combined strategy of chemotherapy, radiation, targeting, and bone marrow transplantation [6]. However, there are side effects commonly associated with leukemia therapy [7]. To avoid or reduce those, it is necessary to develop novel drugs for leukemia treatment.
Many natural and cultivated hybrids of pitcher plant species belonging to Nepenthes (tropical carnivorous plants) are traditionally applied as herbal medicines in Southeast Asia [8]. Nepenthes extracts were reported to inhibit bacterial and fungal growth [9] and inflammation [10]. Different Nepenthes extracts showed antiproliferation effects in several cancer cells, but their effects were rarely reported for leukemia cells. For example, the methanol extract of N. alata Blanco can inhibit breast cancer cell proliferation [11]. Similarly, our previous findings showed that ethyl acetate extracts of N. thorelii × (ventricosa × maxima) (EANT) induced antiproliferation and reactive oxygen species (ROS) in breast cancer cells [12]. However, the potential antiproliferation effect of EANT on leukemia cells remained uninvestigated.
Many ROS-modulating agents were developed to induce oxidative stress in anticancer therapy by excessing the ROS tolerance in cancer cells [13][14][15][16][17]. The rationale is that drug-induced excessive oxidative stress frequently triggers DNA damages [18] and apoptosis [19]. In addition, antioxidants exhibiting dual functions for regulating cellular oxidative stress have been reported [20]. Therefore, a high concentration of antioxidants may induce oxidative stress. However, the antioxidant effect of EANT remains unclear.
The objectives of the present study are to assess the antioxidant ability and antiproliferation effects of EANT on leukemia cells. In addition, the detailed mechanism for anti-leukemia of EANT is also explored in terms of viability, apoptosis, oxidative stress, and DNA damage detection.

EANT Extraction and Chemicals
The extracting condition, detailed high-performance liquid chromatography (HPLC) fingerprint information, and bioactive compounds of EANT were reported previously [12]. Briefly, the air-dried aerial parts of N. thorelii × (ventricosa × maxima) were immersed in methanol and subsequently partitioned by water/ethyl acetate. Finally, the ethyl acetate layer was collected and called EANT further on.

Statistical Analysis
Using JMP 12 software (SAS Campus Drive, Cary, NC, USA), the significance was assessed by one-way analysis of variance (ANOVA) with HSD post hoc test in multiple comparisons. Data showing different alphabets at the top reveal significant differences.

EANT Decreases Cell Viability of Leukemia Cells
According to Figure 2, the cell viabilities (%) of leukemia cells (HL-60, K-562, and MOLT-4) were decreased upon EANT treatment at 24, 48, and 72 h. In addition, the function of oxidative stress and apoptosis in inhibiting the proliferation of EANT in leukemia cells was assessed by their inhibitors, such as NAC and ZVAD. At 24 h of treatment with 4 µg/mL EANT, all antiproliferation effects of EANT on the three leukemia cell lines were converted to normal proliferation by NAC and moderately reverted by ZVAD. . For multiple comparisons, data without the sam letters reveal a significant difference (p < 0.05-0.0001). For example ( Figure 1C), the • OH radica scavenging (%) at 250 and 500 μg/mL EANT show "b and b" indicating nonsignificant difference between each other because they overlap with the same lower-case letters. Similarly, the • OH radica scavenging (%) at 50 and 250 μg/mL EANT showing "d" and "b" indicate significant difference among each other.

EANT Decreases Cell Viability of Leukemia Cells
According to Figure 2, the cell viabilities (%) of leukemia cells (HL-60, K-562, and MOLT-4) were decreased upon EANT treatment at 24, 48, and 72 h. In addition, the func tion of oxidative stress and apoptosis in inhibiting the proliferation of EANT in leukemia cells was assessed by their inhibitors, such as NAC and ZVAD. At 24 h of treatment with 4 μg/mL EANT, all antiproliferation effects of EANT on the three leukemia cell lines were converted to normal proliferation by NAC and moderately reverted by ZVAD.
Moreover, the cytotoxic effect of the main isolated compound plumbagin in EANT [12] was determined on these three leukemic cell lines. The cell viabilities (%) of leukemia cells (HL-60, K-562, and MOLT-4) were decreased upon compound treatment at 24, 48 and 72 h ( Figure 2C). These results suggested that plumbagin may participate in the anti proliferation effects of EANT in leukemia cells.   Figure 3A demonstrates the histograms for cell cycle distribution in EANT-treated leukemia HL-60 cells. In Figure 3B, EANT dose-dependently causes more subG1 and G1 populations than the control in leukemia HL-60 cells. Since 4 μg/mL EANT showed a dra- Moreover, the cytotoxic effect of the main isolated compound plumbagin in EANT [12] was determined on these three leukemic cell lines. The cell viabilities (%) of leukemia cells (HL-60, K-562, and MOLT-4) were decreased upon compound treatment at 24, 48, and 72 h ( Figure 2C). These results suggested that plumbagin may participate in the antiproliferation effects of EANT in leukemia cells. Figure 3A demonstrates the histograms for cell cycle distribution in EANT-treated leukemia HL-60 cells. In Figure 3B, EANT dose-dependently causes more subG1 and G1 populations than the control in leukemia HL-60 cells. Since 4 µg/mL EANT showed a dramatic induction of the subG1 population (an apoptosis-like status), this concentration was chosen to apply to the following time-course experiments to explore the mechanisms involving oxidative stress, apoptosis, and DNA damage.

EANT Increases Populations for SubG1 and G1 Phases in Leukemia Cells
ZVAD on cell viability. Cells (HL-60, K-562, and MOLT-4) were pretreated with NAC (5 mM, 1 h), ZVAD (100 μM, 2 h), or instead post-treated with EANT (0 and 4 μg/mL for 24 h). The 0 μg/mL solution contained no EANT but contained 0.1% DMSO. (C) MTS assay at 24, 48, and 72 h for the main isolated compound plumbagin in EANT. Data, mean ± SD (n = 3). Data showing different alphabets at the top revealed a significant difference (p < 0.0001 for multiple comparisons). In the example of HL-60 cells ( Figure 2B), the EANT 0, NAC/EANT 0, ZVAD/EANT 0, and NAC/EANT 0 show "a", indicating nonsignificant differences between each other because they overlap with the same lower-case letters. Similarly, the NAC/EANT 0, ZVAD/EANT 4, and EANT 4 showing "a, b, and c" indicate significant differences among each other. Figure 3A demonstrates the histograms for cell cycle distribution in EANT-treated leukemia HL-60 cells. In Figure 3B, EANT dose-dependently causes more subG1 and G1 populations than the control in leukemia HL-60 cells. Since 4 μg/mL EANT showed a dramatic induction of the subG1 population (an apoptosis-like status), this concentration was chosen to apply to the following time-course experiments to explore the mechanisms involving oxidative stress, apoptosis, and DNA damage.

EANT Caused Apoptosis in Leukemia Cells
The subG1 increasing effect, as shown in Figure 3, indicates an apoptosis-like response. To further validate the apoptosis effect of EANT, an annexin V/7AAD analysis was performed. Figure 4A demonstrates the annexin V histograms for EANT-treated leukemia HL-60 cells. In Figure 4B, leukemia HL-60 cells following EANT treatment exhibit higher annexin V (+) cells than the control in a dose-dependent manner.
Since NAC recovered EANT-induced antiproliferation (Figure 2), we assessed the NAC effect on apoptosis in leukemia cells. Figure 4C shows the annexin V histograms for EANT-treated leukemia cells with and without NAC pretreatment. In Figure 4D, leukemia HL-60 cells cause more annexin V (+) populations than the control at various time intervals, which is suppressed by NAC.
Cas 3/7 activity assays were further applied to detect the expected caspase activity of apoptosis. In Figure 4E, the caspases 3/7 activities of HL-60, K-562, and MOLT-4 cells are upregulated by EANT at 24 h of treatment, and they are suppressed by NAC pretreatment. In Figure 4F, EANT shows higher c-PARP and c-Cas 3 expressions than the control in a Western blot analysis of leukemia cells. The apoptosis protein c-PARP and c-Cas 3 expressions were suppressed by NAC and ZVAD pretreatment, especially at 24 h of EANT treatment.

EANT Caused ROS Induction in Leukemia Cells
The contribution of oxidative stress in EANT-treated leukemia cells was investigated by ROS monitoring. Figure 5A shows the ROS histograms for EANT-treated leukemia HL-60 cells. Figure 5B shows that leukemia HL-60 cells following EANT treatment exhibit more ROS (+) populations than the control in a dose-dependent manner.
apoptosis. In Figure 4E, the caspases 3/7 activities of HL-60, K-562, and MOLT-4 cells are upregulated by EANT at 24 h of treatment, and they are suppressed by NAC pretreatment. In Figure 4F, EANT shows higher c-PARP and c-Cas 3 expressions than the control in a Western blot analysis of leukemia cells. The apoptosis protein c-PARP and c-Cas 3 expressions were suppressed by NAC and ZVAD pretreatment, especially at 24 h of EANT treatment.

EANT Caused ROS Induction in Leukemia Cells
The contribution of oxidative stress in EANT-treated leukemia cells was investigated by ROS monitoring. Figure 5A shows the ROS histograms for EANT-treated leukemia Since NAC recovered EANT-induced antiproliferation (Figure 1), the NAC effect on ROS induction in leukemia cells was assessed. Figure 5C demonstrates the ROS histograms for EANT-treated leukemia cells with and without NAC pretreatment. In Figure 5D, leukemia HL-60 cells cause more ROS (+) populations than the control at various time intervals, which is suppressed by NAC.

EANT Causes Superoxide Induction in Leukemia Cells
The involvement of oxidative stress of leukemia following EANT was addressed by MitoSOX monitoring. Figure 6A demonstrates the MitoSOX histograms for EANT-treated leukemia HL-60 cells. In Figure 6B, leukemia HL-60 cells following EANT treatment exhibit more MitoSOX (+) populations than the control in a dose-dependent manner.
Since NAC recovered EANT-induced antiproliferation (Figure 1), the NAC effect on MitoSOX induction in leukemia cells was assessed. Figure 6C demonstrates the Mi-toSOX histograms for EANT-treated leukemia cells with or without NAC pretreatment. In Figure 6D, leukemia HL-60 cells cause more MitoSOX (+) populations than the control at various time intervals, which is suppressed by NAC. more ROS (+) populations than the control in a dose-dependent manner.
Since NAC recovered EANT-induced antiproliferation (Figure 1), the NAC effect on ROS induction in leukemia cells was assessed. Figure 5C demonstrates the ROS histograms for EANT-treated leukemia cells with and without NAC pretreatment. In Figure  5D, leukemia HL-60 cells cause more ROS (+) populations than the control at various time intervals, which is suppressed by NAC.

EANT Causes Superoxide Induction in Leukemia Cells
The involvement of oxidative stress of leukemia following EANT was addressed by MitoSOX monitoring. Figure 6A demonstrates the MitoSOX histograms for EANT-treated leukemia HL-60 cells. In Figure 6B, leukemia HL-60 cells following EANT treatment exhibit more MitoSOX (+) populations than the control in a dose-dependent manner.
Since NAC recovered EANT-induced antiproliferation (Figure 1), the NAC effect on MitoSOX induction in leukemia cells was assessed. Figure 6C demonstrates the MitoSOX histograms for EANT-treated leukemia cells with or without NAC pretreatment. In Figure  6D, leukemia HL-60 cells cause more MitoSOX (+) populations than the control at various time intervals, which is suppressed by NAC.

EANT Causes MMP Dysfunction in Leukemia Cells
The function of oxidative stress of leukemia following EANT was addressed by MMP monitoring. Figure 7A demonstrates the MMP histograms for EANT-treated leukemia HL-60 cells. In Figure 7B, leukemia HL-60 cells following EANT treatment exhibit more MMP (−) populations than the control in a dose-dependent manner.

EANT Causes MMP Dysfunction in Leukemia Cells
The function of oxidative stress of leukemia following EANT was addressed by MMP monitoring. Figure 7A demonstrates the MMP histograms for EANT-treated leukemia HL-60 cells. In Figure 7B, leukemia HL-60 cells following EANT treatment exhibit more MMP (−) populations than the control in a dose-dependent manner.

EANT Causes Antioxidant Gene Expressions in Leukemia Cells
The antioxidant system is modulated by oxidative stress [41]. mRNA expressions for antioxidant genes [38,42,43], including NFE2L2, CAT, TXN, HMOX1, and NQO1, were monitored by real-time RT-PCR analysis following EANT treatment in HL-60 cells. These antioxidant genes were upregulated by EANT (Figure 8).  Since NAC recovered EANT-induced antiproliferation (Figure 1), the NAC effect on MMP dysfunction in leukemia cells was assessed. Figure 7C demonstrates the MMP histograms for EANT-treated leukemia cells with and without NAC pretreatment. In Figure 7D, leukemia HL-60 cells cause more MMP (−) populations than the control at various time intervals, which is suppressed by NAC.

EANT Causes γH2AX Type of DNA Damages in Leukemia Cells
γH2AX is a DNA damage marker for double-strand breaks. The change in the DNA damage of leukemia following EANT was addressed by γH2AX monitoring. Figure 9A shows the γH2AX histograms for EANT-treated leukemia HL-60 cells. In Figure 9B, leukemia HL-60 cells following EANT treatment exhibit more γH2AX (+) populations than the control in a dose-dependent manner.

EANT Causes γH2AX Type of DNA Damages in Leukemia Cells
γH2AX is a DNA damage marker for double-strand breaks. The change in the DNA damage of leukemia following EANT was addressed by γH2AX monitoring. Figure 9A shows the γH2AX histograms for EANT-treated leukemia HL-60 cells. In Figure 9B, leukemia HL-60 cells following EANT treatment exhibit more γH2AX (+) populations than the control in a dose-dependent manner.
Since NAC recovered EANT-induced antiproliferation (Figure 1), the NAC effect on the γH2AX type of DNA damage in leukemia cells was assessed. Figure 9C demonstrates the γH2AX histograms for EANT-treated leukemia cells with and without NAC pretreatment. In Figure 9D, leukemia HL-60 cells cause more γH2AX (+) populations than the control at various time intervals, which is suppressed by NAC.

EANT Causes 8-OHdG Type of DNA Damages in Leukemia Cells
8-OHdG is a marker for oxidative DNA damage. The change in the oxidative DNA damage of leukemia following EANT was addressed by 8-OHdG monitoring. Figure 10A demonstrates the 8-OHdG histograms for leukemia cells for EANT-treated leukemia HL-60 cells. In Figure 10B, leukemia HL-60 cells following EANT treatment develop more 8-OHdG (+) populations than the control in a dose-dependent manner.
Since NAC recovered EANT-induced antiproliferation (Figure 1), the NAC effect on the 8-OHdG type of DNA damage in leukemia cells was assessed. Figure 10C demonstrates the 8-OHdG histograms for EANT-treated leukemia cells with and without NAC pretreatment. In Figure 10D, leukemia HL-60 cells cause more 8-OHdG (+) populations than the control at various time intervals, which is suppressed by NAC. Since NAC recovered EANT-induced antiproliferation (Figure 1), the NAC effect on the γH2AX type of DNA damage in leukemia cells was assessed. Figure 9C demonstrates the γH2AX histograms for EANT-treated leukemia cells with and without NAC pretreatment. In Figure 9D, leukemia HL-60 cells cause more γH2AX (+) populations than the control at various time intervals, which is suppressed by NAC.

EANT Causes 8-OHdG Type of DNA Damages in Leukemia Cells
8-OHdG is a marker for oxidative DNA damage. The change in the oxidative DNA damage of leukemia following EANT was addressed by 8-OHdG monitoring. Figure 10A demonstrates the 8-OHdG histograms for leukemia cells for EANT-treated leukemia HL-60 cells. In Figure 10B, leukemia HL-60 cells following EANT treatment develop more 8-OHdG (+) populations than the control in a dose-dependent manner.

Discussion
The anticancer effect of EANT, ethyl acetate extract of N. thorelii x (ventricosa x maxima), has only been reported in breast cancer cells [12], and its antioxidant ability and anticancer effect on leukemia cells remain unclear. In the present study, we assessed the antioxidant ability using five standard antioxidant assays, antiproliferation by MTS assay, and detailed mechanisms by flow cytometry and Western blotting analysis. Several connections between each finding for EANT-treated leukemia cells are discussed below.
As yet, anti-leukemia reports for Nepenthes extracts are rare according to a PubMed search. AML was developed from myeloid cells, while ALL was developed from different types of lymphocytes (B-or T-cells). Both AML and ALL types of leukemia cells may not be metabolically homogeneous and exhibit different metabolic characteristics [44]. For example, AML and T-ALL cell lines show higher glycolytic and cell respiration gene expressions than those of B-ALL cell lines [45]. It is, therefore, interesting to examine the drug responses to EANT on AML (HL-60 and K-562) and ALL (MOLT-4) cell lines. In the current study, IC50 values of EANT-treated AML HL-60 and K-562 and ALL MOLT-4 types of leukemia cells were 3.85, 3.68, and 3.73 μg/mL for the 24 h MTS assay, 1.28, 1.76, and 0.94 μg/mL for the 48 h MTS assay, and 0.96, 1.76, and 0.99 for the 72 h MTS assay, respectively. The time-dependent cytotoxicity of EANT shows at HL-60 cells from 24 to 72 h and at K-562 and MOLT-4 cells from 24 to 48 h. For breast cancer MCF7 and SKBR3 cells, their IC50 values of EANT were 15 and 25 μg/mL following a 24 h MTS assay [12]. Accordingly, leukemia cells showed about 3.5-6.5 fold higher sensitivity to EANT than breast cancer cells did.

Discussion
The anticancer effect of EANT, ethyl acetate extract of N. thorelii x (ventricosa x maxima), has only been reported in breast cancer cells [12], and its antioxidant ability and anticancer effect on leukemia cells remain unclear. In the present study, we assessed the antioxidant ability using five standard antioxidant assays, antiproliferation by MTS assay, and detailed mechanisms by flow cytometry and Western blotting analysis. Several connections between each finding for EANT-treated leukemia cells are discussed below.
As yet, anti-leukemia reports for Nepenthes extracts are rare according to a PubMed search. AML was developed from myeloid cells, while ALL was developed from different types of lymphocytes (B-or T-cells). Both AML and ALL types of leukemia cells may not be metabolically homogeneous and exhibit different metabolic characteristics [44]. For example, AML and T-ALL cell lines show higher glycolytic and cell respiration gene expressions than those of B-ALL cell lines [45]. It is, therefore, interesting to examine the drug responses to EANT on AML (HL-60 and K-562) and ALL (MOLT-4) cell lines. In the current study, IC 50 [12]. Accordingly, leukemia cells showed about 3.5-6.5 fold higher sensitivity to EANT than breast cancer cells did.
The bioactive compounds of EANT, including plumbagin, cis-isoshinanolone, and quercetin 3-O-(6"-n-butyl β-D-glucuronide), were reported previously by high-performance liquid chromatography (HPLC) analysis [12]. Except for cis-isoshinanolone [46], quercetin 3-O-(6"-n-butyl β-D-glucuronide) exhibits anticancer effects on liver and breast cancer cells [47], and plumbagin shows anticancer effects on several types of cancer cells [48]. For the ALL type of leukemia cells (MOLT-4), plumbagin shows an IC 50 value of 0.19 µg/mL for the 24 h CCK-8 assay, but it shows no cytotoxicity to normal peripheral blood mononuclear cells [49]. For the AML type of leukemia cells (Kasumi1 and HL-60), plumbagin shows IC 50 values of 0.85 and 0.28 µg/mL for the 24 h CCK-8 assay [50] and the 48 h MTT assay [51]. Similarly, plumbagin shows IC 50  Plumbagin targets the transactivation domain of Myb to suppress Myb activity [52]. In addition, five potential targets of plumbagin were reported, namely phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3Kγ), AKT1, Bcl-2, nuclear factor kappa B subunit (NF-κB), and signal transducer and activator of transcription 3 (STAT3), using molecular docking and (un)binding simulation analysis [53]. Making use of the differentiation-inducing effect is a promising strategy for the treatment of AML. Plumbagin also shows a differentiationinducing effect on AML HL-60 cells [52]. Moreover, plumbagin can inhibit the proliferation of primary AML cells derived from patients but not for normal hematopoietic progenitors. Therefore, further evaluation of the differentiation-inducing effects of EANT and other EANT-derived compounds on leukemia cells is warranted in the future.
For cellular redox homeostasis, exogenous antioxidants may provide a bi-functional regulation of oxidative stress. They decrease oxidative stress at physiological concentrations but increase oxidative stress at high concentrations [20]. Similarly, ROS-inducing agents [31], natural products, and herbal medicines [54,55] show anticancer effects by generating exogenous ROS to exceed the tolerance of redox homeostasis in cancer cells [56].
The oxidative stress-inducing effect of EANT was validated by the evidence of ROS, MitoSOX, and MMP flow cytometry in leukemia HL-60 cells (Figures 5-7). Moreover, mitochondrial metabolism is a vital target for AML therapy [59]. Targeting mitochondria for ROS and MitoSOX modulations can improve the therapeutic effects of AML [23,60]. Similarly, natural products, such as Rosa cymose fruits, exhibit DPPH antioxidant ability and have ROS-inducing potential in leukemia cells [23]. Some marine sponge-derived natural products also demonstrate both an antioxidant ability and antiproliferation of cancer cells [61,62].
Antioxidant gene expression and oxidative stress have a cross-talk interaction [63,64]. In response to sustained exogenous oxidative stress, NFE2L2 and its target TXN were activated [65]. CAT was also triggered by oxidative stress [66]. CAT and HMOX1 mRNA and protein in mice were upregulated in response to UVC irradiation-induced oxidative stress [67]. Moreover, NQO1 knockdown suppressed oxidative stress in prostate cancer cells [68]. Consistent with the present study, EANT enhanced the mRNA expressions of NFE2L2, CAT, TXN, HMOX1, and NQO1 genes (Figure 8) in leukemia cells in response to EANT-induced oxidative stress. Therefore, the antioxidant capacity of EANT is probably associated with the antiproliferation response to leukemia cells through oxidative stress generation. It is noted that mRNA levels may not be in accordance with the protein levels of antioxidant signaling genes. A detailed investigation of antioxidant protein expressions of leukemia cells following EANT treatment is warranted in the future.
The toxic effect of high oxidative stress frequently induces apoptosis [69] and DNA damage [70] in cancer therapy. For example, the ethanol extract of Rosa cymose fruits upregulates ROS generation, disrupts MMP, induces a γH2AX type of DNA damage, and triggers apoptosis in leukemia cells [23]. As the responses of breast cancer cells [12], EANT also shows cellular (ROS) and mitochondrial (MitoSOX) oxidative stresses to trigger apoptosis and to induce a γH2AX type of DNA damage to leukemia cells. Moreover, oxidative DNA damage adduct 8-OHdG is also induced following EANT treatment.
EANT induces oxidative stress; however, the dependence of oxidative stress on all test changes in leukemia cells following EANT treatment needed to be examined. Using NAC pretreatment, the EANT-associated changes of cell viability, cell cycle dysregulation, cellular and mitochondrial oxidative stress, apoptosis, and DNA damage were reverted. Accordingly, the antiproliferation effect and mechanisms were mediated by oxidative stress in leukemia cells.

Conclusions
The antioxidant and antiproliferation properties have rarely been examined for Nepenthes ethyl acetate extract (EANT) in leukemia cells. In the present study, we firstly reported that EANT exhibits antioxidant abilities and demonstrates the antiproliferation of acute leukemia cells. With the pretreatment of ROS and apoptosis inhibitors, oxidative stress and apoptosis were validated to contribute to the antiproliferation of leukemia cells following EANT exposure. Mechanistically, EANT causes cell cycle disturbance associated with apoptosis expression and signaling and induces several oxidative stress changes and DNA damages in leukemia cells. ROS inhibitors alleviated all these EANT-induced changes. The weakness of the present study was the lack of some experiments on the three leukemia cell lines. Most results were demonstrated only using one leukemia cell type (HL-60) to investigate the possible antiproliferation mechanisms. In conclusion, EANT exhibits antiproliferation and apoptosis function on leukemia HL-60 cells relying on oxidative stress modulation.