Limonin induces apoptosis of HL‐60 cells by inhibiting NQO1 activity

Abstract Limonin is an important bioactive substance in citrus fruits, especially in seeds, which has great potential in cancer prevention and treatment. In order to explore the anticancer activity based on interaction between limonin and NQO1, Human promyelocytic leukemia cells (HL‐60) were studied in vitro. We found that limonin could inhibit proliferation and promote apoptosis of HL‐60 cells, and the effect was positively correlated with its dosage. Western blot results showed that limonin could activate the endogenous apoptosis pathway mediated by mitochondria via up‐regulating pro‐apoptotic proteins (Bax, cytochrome c, Caspase3, and Caspase9) and down‐regulating anti‐apoptotic proteins (Bcl‐2), thus inhibiting the proliferation of HL‐60 cells and promoting apoptosis, which further proved the anticancer activity of limonin from the molecular mechanism. At the same time, limonin down‐regulated the expression of NQO1, indicating that limonin may indirectly act on the apoptosis pathway by regulating the expression activity of antioxidant enzymes in vivo, thus exerting its inhibitory effect on tumor cells, which provides an idea for the molecular mechanism that natural products can indirectly exert their anticancer effect by regulating the activity of antioxidant enzymes.

has always been a research hotspot. Due to the characteristics of citrus natural products such as easy availability and low toxic, researchers hope to use citrus-derived substances to inhibit tumor development (Tian et al., 2001) or mitigate the side effects (Tahaghoghi-Hajghorbani et al., 2019)of chemotherapy drugs.
Cancer is one of the most important leading causes of death.
Cancer causes great psychological and physical damage to people, especially some adolescent cancers, such as juvenile leukemia, will be devastating to the life will and family hope when the blow. Cancer prevention, in addition to avoiding exposure to carcinogens and keeping regular exercise, supplementing with natural products, especially fruits, is also a very important measure (Filocamo et al., 2015).
Limonin (Figure 1), also known as obaculactone and evodin, is a type of secondary metabolites belonging to the tetracyclic triterpenoids, which is usually found in the plants of Rutaceae and Meliaceae. Limonin is extensively distributed in citrus fruits, which is the main cause of citrus bitterness (Emerson, 1948). Limonin has broad-spectrum biological activities, such as anticancer, antiviral, antioxidant, antimicrobial, anti-inflammatory, anti-obesity, liver protection, and anti-atherosclerosis (Higby, 1938, Emerson, 1948, Kelley et al., 2015Lee et al., 2016;Yang et al., 2014). Rahman et al. (2015) found the anticancer effect of limonin on various human cancer cells by MTT method. Their results showed that limonin could significantly inhibit the growth of cancer cells by promoting the expression of apoptosis-related proteins, and the effect was dose dependent. However, it has not been reported whether limonin could inhibit cancer by regulating the activity of antioxidant enzymes. NAD (P)H Quinone Dehydrogenase 1 (NQO1) is an intracellular, cytosolic enzyme which is known to catalyze two-electron reduction of a broad range of substrates. It plays an important role in the detoxification of environmental carcinogenic factors (Ross & Siegel, 2004).
However, although NQO1 protects cells from oxidative stress (Davies et al., 2005), it also has carcinogenic effect inversely. Studies have shown that the expression of NQO1 is up-regulated in many malignant tumors, such as melanoma, pancreatic cancer, intrahepatic cholangiocarcinoma, lung cancer, and breast cancer (Hu et al., 1996;Kelsey et al., 1997;Pan et al., 1995;Ross & Siegel, 2004). The overexpression of NQO1 can induce cell apoptosis and promote the proliferation of cancer cells (Luo et al., 2018). At present, evidence has shown that natural products can exert anticancer activity through inhibiting the expression of NQO1 (D'Anneo et al., 2010;Pink et al., 2000;Zhao & Wu, 2018), but there are few reports on the similar function of active substances in citrus fruits, especially limonin.
Our present study found that limonin can promote the apoptosis of promyelocytic leukemic cell line (HL-60) by inhibiting the activity of NQO1, which provides new evidence and support for the exploration of the bioactivity of limonin and the regulation function of natural products on antioxidant enzyme activity.

| Cell line and cell culture
Human promyelocytic leukemia cells (HL-60) were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese F I G U R E 1 The chemical structure of limonin Academy of Sciences. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 20 mM HEPES, 100 U/ml penicillin, and streptomycin. Cells were maintained at 37°C in a humidified incubator containing 5% CO 2 . Exponentially growing cells were used for further experiments.

| Cell viability assay
Cell viability of HL-6 cells was evaluated by a cck-8 assay according to  with small modifications. Briefly, cells at exponentially growing phase were seeded in flat bottomed 96-well plates at an initial density of 1 × 10 4 cells/well with 200 μl medium.
Limonin and dicoumarol were dissolved in DMSO (DMSO accounted for 2.0% of the final volume, in the ratio of which the limonin solubility was 94.19%, and exerted no significant threat to the cell viability based on our pre-experiment). Cells were treated with serial dilutions of limonin (25, 50, 100, 200, and 400 μM). Dicoumarol with a concentration of 5 μM was set as positive control, and DMSO was set as solvent control. After incubation for 24 hr, the medium was removed and the cells were washed twice with PBS. Cck-8 reagents diluted with FBS-free RPMI 1640 medium was added to the wells and incubated for 1h. The absorbance was detected at 450 and 620 nm by a microplate reader (Synergy H1, BioTek, Winooski, VT, USA). The cell viability was calculated as follows: Cell viability (%) = Treatment (A 450 -A 620 )/Solvent control(A 450 -A 620 ) × 100%. Each experiment was performed in triplicate and repeated three times independently.
Dicoumarol with a concentration of 5 μM was set as positive control, and DMSO was used as solvent control. After treatment, the cells were washed twice with ice-cold PBS. Trypsin (0.25%) / EDTA (0.02%) was applied to dissociated the adherent cells. Both suspension and attached cells were collected after centrifuging at 85 g for 5 min, and the density was adjusted to 1 × 10 3 cells/ml. Apoptotic cells were identified by double supravital staining. For 1 ml cells, 200 μl of Annexin V-FITC was added to the cells followed by the addition of 10 μl of propidium iodide (PI). The samples were stained for 5 min in the dark at room temperature. Then cell apoptosis rate was detected immediately by flow cytometry.

| Quantitative Real-Time PCR assay
The Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed according to our previous report with small modifications . Briefly, cells were plated in 6-well plates with densities of 2 × 10 6 cells per well for HL-60. After 24 hr of culture, the cells were treated with limonin at a concentration of 50-200 μM for 12 hr, respectively, 5 μM diucoumarol was used as positive control. Total RNA isolation, cDNA synthesizing, and Quantitative Real-Time PCR were performed using commercial kits according to the manufacturer's protocols. The primer sequences were listed in Table 1. GAPDH was used as the control, and the relative level of gene expression was calculated using the 2 −ΔΔCt method.
Each experiment was repeated three times independently.

| Statistics
All experiments were repeated at least three times, and data were expressed as the mean ± SEM. SPSS 19.0 software (IBM, Armonk,

| Inhibition activity of limonin on the proliferation of HL-60 cell
To examine the effect of limonin on cell proliferation, HL-6 cells were treated with various concentrations of limonin for 24 hr, and then, the cell viability was measured using a cell counting kit-8 (cck-8) assay. In

| Regulatory activity of limonin on the expression of apoptosis-related genes and proteins in HL-60 cells
In order to investigate the molecular mechanism by which limonin promotes apoptosis of HL-60 cells, we examined the expression lev- Apoptosis is a form of programmed cell death, which could be mediated by members of the Caspase family. One of the most classic apoptosis pathways is the intrinsic mitochondrial pathway.
Bcl-2 protein family play a crucial role in this pathway by regulating mitochondrial membrane potential. Both the anti-apoptotic protein Bcl-2 (B-cell CLL/lymphoma 2) and the pro-apoptotic protein Bax (Bcl2-associated X protein) belong to the Bcl-2 family (Huang et al., 2008;Wang et al., 2015). When the pro-apoptotic protein

| DISCUSS IONS
Limonin is an important secondary metabolite in citrus fruits, especially in seeds (Poulose et al., 2006). It has a variety of biological activities, such as anticancer, antioxidant, anti-inflammatory, neuroprotective, and antibacterial. In recent years, a large number of studies have shown that limonin has great potential in cancer prevention and treatment by scavenging free radicals, stimulating the activity of glutathione transferase, inhibiting the activity of carcinogenic chemicals, and inhibiting the proliferation of tumor cells (Bodduluru et al., 2014;Galal et al., 2015, Gong et al., 2019. NQO1 is a flavoprotein that can catalyze two-electron reduction of a broad range of substrates (Ross & Siegel, 2004). It has a variety of cytoprotective effects, especially in the prevention of cancer and oxidative stress-related diseases (Oh & Park, 2015). However, in recent years, a large amount of evidence showed that NQO1 has a two-sided effect in the occurrence of cancer. It can be used as both a tumor suppressor and a tumor promoter. It has been reported that NQO1 knockout could inhibit the proliferation of glioblastoma cells, while overexpression promoted cell proliferation (Luo et al., 2018).
In addition, the expression level of NQO1 has been found to be abnormally elevated in a variety of solid tumor cells (Oh & Park, 2015).
Studies have shown that the overexpression of NQO1 was induced by overactivation of Nrf2, which helped malignant cells escape from extreme oxidative stress (Menegon et al., 2016). It can be seen that whether NQO1 plays an It can be seen that whether NQO1 plays an anti-tumorigenic or tumorigenic effect depends on different conditions and cell types, which also reflects the potential of NQO1  (Haga et al., 2003;Wang & El-Deiry, 2004). Bcl-2 and Bax genes in the Bcl-2 family are the most important regulators of apoptosis (Krueger et al., 2001). When the cells are internally stimulated, the homeostasis of the cellular environment is out of balance. Then, the mitochondrial membrane potential decreases, and Bax/Bcl-2 proteins are recruited in the mitochondrial outer membrane, which can change the conformation of the mitochondria and form mitochondrial permeability transition pore (mPTP) on the mitochondrial outer membrane. The pro-apoptotic factors such as cytochrome c are promoted to release into the matrix through mPTP, and cytochrome c binds to the apoptotic protease activator Apf-1 to form a complex, inducing caspase-9 precursor to cleave itself into active cleaved-Caspase-9 and further activate downstream cysteine (Caspase3, caspase-7) cascade reaction, and finally induce apoptosis .
Abnormal apoptosis is an important factor in cancer occurrence, so inducing tumor cell apoptosis has become an effective measure for cancer treatment (Kumar et al., 2013). Many studies have found that a lot of natural products can exert anticancer activity by inducing apoptosis of tumor cells (Wang, Zhong, et al., 2018 (Suzuki et al., 1999) in N-(4-hydroxyphenyl) retinamide)-induced apoptosis of cervical cancer cells.
Limonin belongs to triterpenes, and other triterpenoid natural products with similar structure also have important anticancer potential. For example, triterpenes isolated from Forsythia suspensa have obvious inhibitory effect on human digestive tract tumor cells, and the main way to induce apoptosis of SGC-7901 cells may be to regulate the expression of apoptosis-related proteins (Sun & Zhang, 2010). Triterpenes extracted from Pleurotus ferulae (ethylacetate fraction of Pleurotus ferulatus triterpenoid, PFTP-E) can effectively induce apoptosis of Eca109 esophageal cancer cells, and its anti-tumor mechanism is related to mitochondrial damage pathway, cycle arrest, and endoplasmic reticulum stress (Lei et al., 2019).
Ursolic acid can regulate PI3K/AKT/mTOR signal pathway and hinder the development of colorectal cancer (Cui & Su, 2018). These studies show that the mechanism of triterpenoid natural products with different structures on cancer cells is different, and the action pathways of the same natural products on different cancer cells may also have variabilities, which provides a way to further explore the mechanism of limonin in anticancer and to explore the relationship between triterpene natural products with different structures and anticancer function.

| CON CLUS ION
In this study, by using Human promyelocytic leukemia cells (HL-60), we investigated the pro-apoptotic activity of limonin. Limonin inhibited the proliferation and promoted the apoptosis of HL-60 cells in a dose-dependent manner. Mechanism exploration demonstrated that the apoptosis of HL-60 was related to the up-regulation of Bax, cytochrome c, cleaved-Casepase3, and cleaved-Caspase 9 expression, and the down-regulation of NQO1 and Bcl-2 expression. The results showed that limonin may indirectly affect the apoptosis pathway by inhibiting the activity of antioxidant enzyme NQO1, thus promoting the apoptosis of HL-60. Our study may complement the anticancer molecular mechanism of natural products.

ACK N OWLED G M ENTS
This word was funded by the Natural Science Foundation of Zhejiang Province (LQY18C200001).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.