Lung cancer is the most commonly diagnosed cancer among all malignancies in the world and the leading cause of cancer death [1
]. Over 2.2 million cases of lung cancer are diagnosed in 2018, accounting for 11.6% of the total cases, and 1.76 million deaths in 2018 are attributed to lung cancer, accounting for 18.4% of all cancer-related deaths [1
]. The most common type of lung cancer is adenocarcinoma, comprising around 40% of all lung cancer cases [2
]. Despite the advances in understanding this disease mechanism and the development of new therapeutic strategies, lung adenocarcinoma is still one of the most aggressive and rapidly fatal types of cancer [3
]. Thus, new therapeutic agents with high efficiency for this deadly disease are urgently needed.
Reactive oxygen species (ROS) are involved in the regulation of many important cellular processes such as cell cycle and apoptosis [5
]. For example, increased ROS generation in cancer cells activates signaling pathways necessary for the initiation, promotion and progression of tumors, and also contributes to tumor resistance to chemotherapy [8
]. ROS can activate certain specific signaling pathways, including hypoxia-inducible factors (HIFs), phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPKs) cascade kinases [5
]. However, the extremely reactive hydroxyl radical form of ROS generated from hydrogen peroxide can oxidize proteins, lipids and DNA massively, leading to damage or genomic instability [12
]. Cancer cells exhibit higher ROS levels than normal cells [8
]. However, cancer cells have an increased activity of antioxidant enzymes to counteract these ROS levels. Thus, therapeutic strategies that either increase ROS generation and/or decrease antioxidant defense in cancer cells may lead to the activation of various cell death pathways and thus limit the cancer progression [7
, commonly known as white mulberry, belongs to the Moraceae family. In Asian countries, the leaves, fruits and barks of M. alba
have long been used as traditional medicine to protect liver damage, improve eyesight and lower blood pressure [14
]. It possesses many biological activities, including anti-hyperlipidemic, anti-hypertensive, anti-hyperglycemic, anti-microbial, anti-allergic, anti-inflammatory, hepatoprotective and neuroprotective activities [10
]. Phytochemical studies of M. alba
have revealed various constituents, including terpenoids, alkaloids, flavonoids, phenolic acids, stilbenoids and coumarins [14
]. These constituents exert a variety of pharmacological effects, including anti-diabetes [16
], antioxidant [17
], anti-inflammatory [19
], neuroprotective [21
] and anticancer effects [22
]. For example, morusin, a prenylated flavonoid isolated from the root bark of M. alba
, has been shown to inhibit cell proliferation and tumor growth by downregulating c-Myc oncogene in human gastric cancer [23
], and has also been shown to induce apoptosis in human non-small cell lung cancer cells by suppressing EGFR/STAT activation [24
Albanol B (ABN-B) is an arylbenzofuran derivative isolated from M. alba
. ABN-B and has been demonstrated to possess antioxidant [25
], anti-inflammatory [26
], anti-Alzheimer [27
] and anti-diabetes properties [28
]. However, further study is needed to elucidate the anti-cancer effect of this compound and its underlying mechanism. As part our continuing search for an anti-cancer compound from M. alba,
we investigated the anti-cancer activity of ABN-B in lung cancer cell lines. As a result, we found that ABN-B exhibited in vitro and in vivo anti-cancer activity that triggered cell cycle arrest at G2
/M and apoptosis. Moreover, we demonstrated that ABN-B showed active anti-cancer activity by enhancing the mitochondrial ROS production in human lung cancer cells.
Despite the substantial progress that has been made in the treatment of lung cancer, lung cancer remains the most lethal type of cancer worldwide, accounting for more than 1.7 million deaths each year [1
]. In the present study, we found that ABN-B, an arylbenzofuran derivative from M. alba
, exerts an anti-cancer effect in vitro and in vivo models of lung cancer cells by inducing cell cycle arrest at G2
/M and apoptosis in lung cancer cells. Mechanistically, ABN-B leads to the activation of the PI3K/AKT and ERK1/2 MAPK pathways via the production of mitochondrial ROS, which resulted in the induction of apoptosis and cell cycle arrest at G2
/M in human lung cancer cells (Figure 8
E). To our knowledge, this is the first report to demonstrate the anti-cancer effect in vitro and in vivo of ABN-B in lung cancer cells.
Natural products have long and rich experience in treatment of cancer and are accepted increasingly as complementary and alternative therapy [31
]. Anticancer drugs discovered from natural products have been used clinically for cancer treatment as the conventional anticancer drugs [30
]. Extract of M. alba
has been shown to exert anticancer effects on various human cancer cell lines. For example, extract of M. alba
root bark induces cell growth arrest and apoptosis in SW480 human colorectal cancer cells by activating ATF3 expression and down-regulating cyclin D1 level [32
], and also reduces the viability of multidrug-resistant MCF-7/Dox cells by inhibiting YB-1-dependent MDR1 expression [33
]. Furthermore, many compounds with a variety of antitumor mechanisms have been identified from M. alba
. For example, one study has indicated that mulberrofurans, moracins, sanggenon O and albafuran A inhibit hypoxia-inducible factor-1 accumulation and hypoxia-induced vascular endothelial growth factor secretion in Hep3B cells [34
]. Mulberrofuran G has been shown to induce apoptotic cell death in HL-60 cells via both the cell death receptor pathway and the mitochondrial pathway [35
]. In the present study, we showed that ABN-B exerted an anti-proliferative effect on human lung cancer cells by inducing cell cycle arrest at G2/M and apoptosis. Moreover, ABN-B dose-dependently suppressed Ex-3LL tumor growth in a syngeneic implantation model, indicating that ABN-B could be considered as a new lead compound for the development of anti-cancer agent against lung cancer. Moreover, extract of M. alba
could be of value for further exploration as a potential anti-cancer agent for the treatment of lung cancer.
ROS plays a dual role in cancer [12
]. On the one hand, ROS can promote protumorigenic signaling, facilitating the proliferation, survival and adaptation to hypoxia of cancer cells. On the other hand, ROS can promote anti-tumorigenic signaling and trigger oxidative stress–induced cancer cell death. Cancer cells have an increased ROS level compared with normal cells due to their high metabolic rate and mitochondrial dysfunction, which leads to an increased susceptibility to oxidative stress [8
]. Thus, ROS can eventually increase beyond a certain threshold level that is incompatible with cellular survival, resulting in oxidative stress-induced cell death. Several natural products that increase cellular ROS levels have been shown to selectively target cancer cells [36
]. For example, dietary phytochemicals such as polyphenols, flavonoids, and stilbenes have the capacity to inhibit cancer cell proliferation and induce apoptosis and autophagy [36
]. For example, piperine, the most abundant alkaloid found in Piper longum
, suppresses tumor growth in vitro and in vivo by inducing cell cycle arrest at G2
/M and apoptosis via ROS production [38
]. In the present study, we demonstrated that ABN-B increased the productions of both intracellular and mitochondrial ROS, and that co-treatment with mito-TEMPO, a specific scavenger of mitochondrial ROS, suppressed ABN-induced cell cycle arrest at G2
/M and apoptosis, indicating that ABN-B has an anti-proliferative effect in human lung cancer cells by inducing mitochondrial ROS production.
Research has shown that ERK activity can promote apoptotic pathways, cell cycle arrest or autophagic vacuolization [40
]. These effects require sustained ERK activity in specific subcellular compartments and could depend on the presence of reactive oxygen species [40
]. Studies have shown that ROS promotes sustained ERK activation by promoting the activation of tyrosine kinase receptors and by inhibiting ERK-directed phosphatases such as DUSP1 and DUSP6 [41
]. The AKT signaling pathway has also been implicated in sensitizing cells to apoptosis, and the ROS-mediated activation of AKT induces apoptosis in prostate cancer cells [45
]. For example, piroxicam, a traditional non-steroidal anti-inflammatory drug, causes apoptosis by ROS-mediated AKT activation, and artocarpin, an isoprenyl flavonoid, induces apoptosis via the ROS-mediated activation of MAPKs and AKT in non-small cell lung cancer cells [47
]. In the present study, we demonstrated that ABN-B induced the phosphorylation of ERK1/2 and AKT in human lung cancer cells, but not of JNK and p38 MAPKs, via mitochondrial ROS production. These two pathways appeared to be involved in both ABN-B-induced cell cycle arrest and apoptosis. Co-treatment of U0126, a specific MEK inhibitor, inhibited the ABN-B-induced up-regulation of PARP cleavage as well as the down-regulation of Bcl-2 and cyclin B1 expressions, but not of CDK1. Co-treatment of LY294002, a specific PI3K inhibitor, also inhibited the ABN-B-induced up-regulation of PARP cleavage and down-regulation of Bcl-2, suggesting that both of the ERK-1/2 and AKT pathways might be involved in the induction of ABN-B-mediated apoptosis. However, LY294002 inhibited the ABN-B-induced down-regulation of CDK1 expression, but not that of cyclin B1, suggesting that the ERK and AKT pathways could regulate the ABN-induced down-regulation of cyclin B1 and CDK1 expressions, respectively.
In summary, the present study demonstrated for the first time that ABN-B, an arylbezofuran derivative from M. alba, exerts anti-cancer in vitro and in vivo in lung cancer cells which are associated with mitochondrial ROS production and the subsequent activation of ERK and AKT, suggesting that the ABN-B-induced mitochondrial ROS production could be an important mechanism for its anti-cancer effect. These study findings provide a rational basis for the usage of M. alba extracts for cancer treatment in traditional oriental medicine. In addition, ABN-B might be a valuable compound meriting further research as an anti-cancer agent. However, the precise mechanisms by which ABN-B increases mitochondrial ROS production must be further elucidated to better understand the anti-cancer activity of ABN-B.
4. Materials and Methods
4.1. Cell Culture
A549, BZR, H1975 and H226 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). A549, H1975 and H226 cells were maintained in RPMI 1640 medium, while BZR cells were maintained in DMEM. A murine Ex-3LL (Lewis lung carcinoma) cell line, which is derived from 3LL, was purchased from JCRB Cell Bank (Osaka, Japan) and maintained in RPMI 1640 medium. All media were supplemented with penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% heat-inactivated FBS (Hyclone, Logan, UT, USA) and all cells were cultured in a humidified chamber with a 5% CO2 atmosphere at 37 °C.
4.2. Chemicals and Reagents
Anti-caspase-7, anti-Akt, anti-phospho-Akt (S473), anti-p38, anti-phospho-p38, anti-phospho-ERK1/2, anti-ERK1/2, anti-PARP (Poly [ADP-Ribose] Polymerase), anti-JNK (c-Jun N-terminal kinase) and anti-phospho-JNK antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-CDK1, anti-Bcl-2, anti-Cyclin B1, and anti-p21Waf1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), NAC (N-acetyl cysteine), and anti-α-tubulin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). LY294002 and U0126 were obtained from Calbiochem (San Diego, CA, USA). Mito-TEMPO was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
4.3. Isolation of ABN-B
ABN-B was obtained from the twigs of M. alba
. Briefly, the air-dried powdered root bark of M. alba (10 kg) was extracted with methanol under reflux and then filtered. The methanol extract was concentrated under reduced pressure to give a residue (1.2 kg), which was suspended in distilled water and successively partitioned with n-hexane, dichloromethane and ethyl acetate. The ethyl acetate fraction (304.3 g) was fractionated by extensive column chromatography (CC) with silica gel eluted by a gradient of 0→100% methanol in dichloromethane to afford twenty fractions (F1–F20). Fraction F5 (6.8 g) was fractionated on silica gel CC eluted with dichloromethane-methanol (20:1 to 0:1, v
) to afford 16 sub-fractions (F5.1–F5.16). Sub-fraction F5.15 (623 mg) was passed over an RP-C18
silica gel column using methanol-water system (2:1, v
) as a mobile phase to yield ABN-B (150 mg). The structure and purity of ABN-B were confirmed by 1
H and 13
C nuclear magnetic resonance spectra (Supplementary Figures S2 and S3
4.4. Cell Viability and Proliferation Assays
MTT-based colorimetric assay was used to measure the cytotoxic effect of ABN-B. In brief, cells were seeded in 96-well plates (3 × 104 cells per well) and allowed to grow to the plate for 24 h. ABN-B was then treated to the wells at different concentrations and the plates were further incubated for 24 h or 48 h. At the end of incubation, 20 µL of MTT solution (5 mg/mL) was added to each well and was further incubated for 4 h. The concentrations needed to reduce the cell density by 50% (IC50 values) were calculated through non-linear regression analysis. Cell proliferation was measured using the BrdU Cell Proliferation Assay Kit (Cell Signaling Technology, Danvers, MA, USA) and performed following the manufacturer’s instructions.
4.5. Colony Formation Assay
Colony formation assay was performed as described previously [49
]. Briefly, 500 cells per well were seeded in 12-well plates and allowed to adhere to the plate for 24 h, and then ABN-B was added to the wells at different concentrations. Following 48 h incubation, the medium was replaced with fresh medium with or without ABN-B, and the cells were further grown for an additional five days. After the cells were washed with PBS (pH 7.4) twice, the cells were stained with crystal violet solution (0.1%) diluted in ethanol 40% for 5 min. Then, the stain solution was removed using tap water and the cells were air-dried and finally, the number of colonies was counted.
4.6. Annexin V/PI Double Staining
The quantification of cell death was evaluated by flow cytometry using an Annexin V-FITC apoptosis detection kit according to the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA). The cells were treated with the indicated concentrations of compounds for either 24 h or 48 h, then were collected by centrifugation. Cell pellet was stained with Annexin V-FITC and propidium iodide (PI) in a binding buffer for 15 min at room temperature in the dark. Annexin V-FITC/PI stained cells were analyzed by flow cytometry (FACS Calibur, Becton-Dickinson, San Jose, CA, USA).
4.7. Western Blot Analysis
For the preparation of whole cell lysates, the cells were lysed in a lysis buffer (50 mM Tris-HCl [pH 7.5], 1% Nonidet P-40, 1 mM EDTA, and 150 mM NaCl) in the presence of protease inhibitor cocktail (Sigma-Aldrich) and 5 mM sodium orthovanadate. The lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blot analysis. Western blots were incubated overnight with the indicated antibodies (1:1000 dilution) and anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (1:2000 dilution) were used to visualize signals using an enhanced chemiluminescence system (ThermoFisher Scientific, Rockford, IL, USA), and the band intensity was quantified using Image J software (NIH, Bethesda, MD, USA).
4.8. Cell Cycle Distribution Analysis
For the determination of cell-cycle distribution, the cells were treated with the indicated concentrations of ABN-B for 48 h, and then washed and centrifuged. The pellets were fixed in ice-cold 80% (v/v) ethanol for 1 h at 4 °C. The cells were subsequently centrifuged and incubated with cold PI solution (50 µg/mL PI and 0.1 mg/mL RNase A for 30 min in the dark. Cell cycle distribution were analyzed using FACSCalibur (Becton Dickinson, San Jose, CA, USA).
4.9. Measurement of Intracellular and Mitochondrial ROS Level
2′,7′-Dichlorofluorescin diacetate (DCF-DA, Sigma-Aldrich) and MitoSOX red (Molecular Probes, Invitrogen, Carlsbad, CA, USA) were used to measure the levels of intracellular ROS and mitochondrial ROS, respectively, as described previously [50
]. Cells were pretreated with NAC (100 µM) or Mito-TEMPO (50 µM) for 30 min, followed by the treatment with either the indicated concentrations of ABN-B for 2 h or 30 µM of ABN-B for the indicated period of time. The cells were then treated with either DCF-DA (10 µM) or MitoSOX (5 µM) in the dark for 30 min, and harvested after washing with PBS. Their fluorescence intensities were assessed using a FACS Calibur flow cytometer (Becton-Dickinson, for DCF-DA, excitation wavelength: 485 nm, emission wavelength: 535 nm; for MitoSOX, excitation wavelength: 510 nm, emission wavelength: 580 nm).
4.10. In Vivo Tumor Growth Assay and Detection of Apoptosis in Tumor Tissues
All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (IACUC approval No. KW-190403-1, approval date 9 April 2019). For in vivo tumor growth assay, Ex-3LL cells (1 × 107 cells per mouse) were subcutaneously injected into the right flank of 4-week-old male C57BL6 mice (Orient Bio Inc., Seongnam-si, Korea). Tumor growth was monitored by caliper measurements and tumor volume was calculated as the following formula: W2 × L × 0.5, where W is the short diameter of the tumor, L is the long diameter of the tumor. Treatment was started when tumor volumes reached approximately 100 mm3. Mice (5 mice/group) were injected intraperitoneally with ABN-B (50 or 100 mg/kg) dissolved in dimethyl sulfoxide:chremophore-EL:PBS (1:1:8 by volume) or control vehicle three times a week. The mice were sacrificed after 21 days, and tumors were removed and embedded in OCT compound. Tissue samples were sectioned at a thickness of 5 μm. For antigen retrieval, the slides were pretreated by heating at 95 °C for 5 min in a retrieval buffer (10 mM Tris-HCl, 1 mM EDTA, 0.05% Tween 20, pH 8.0). Apoptosis in tumor tissues was detected using TdT DAB In situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD, USA).
4.11. Statistical Analysis
The results are presented as the mean ± standard error of the mean (SEM). Statistical significance was conducted using one-way analysis of variance (ANOVA) and the differences between the experimental groups were further compared by Fisher’s least significant difference test.