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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 4 | Page : 454-460
Brusatol inhibits proliferation, migration, and invasion of nonsmall cell lung cancer PC-9 cells
Lu-Ming Yang1, Wen-Min Zhou1, Qiao-Ru Guo1, Xin-Yue Fan1, Dong-Yu Huang1, Xiao-Fei Sun2, Jie Yuan2, Hua Yu3, Hu-Biao Chen4, Jian-Ye Zhang1
1 Key Laboratory of Molecular Target and Clinical Pharmacology and The State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences and The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
2 Department of Pharmaceutical Sciences, Xinjiang Second Medical College, Karamay, China
3 Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao, China
4 School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China
Correspondence Address:
Dr. Hu-Biao Chen
School of Chinese Medicine, Hong Kong Baptist University, Hong Kong
China
Dr. Jian-Ye Zhang
Key Laboratory of Molecular Target and Clinical Pharmacology and The State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences and The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.353662
Objective: The purpose of this study was to investigate the inhibitory effect of brusatol, a nigakilactone extracted from Brucea javanica, on lung cancer for development of therapeutic drugs. We explored the effects of brusatol on the proliferation, migration, and invasion of lung cancer PC-9 cells in vitro and analyzed the mechanisms involved. Materials and Methods: MTT assay was used to determine the effect of brusatol on the proliferative capacity of PC-9 and H1975 cells in vitro. The half-maximal inhibitory concentrations (IC50) were calculated and used as a reference for subsequent experiments. Variations in the number and size of tumor cell clusters were monitored by the colony formation assay as evidence for the effect of brusatol on cell proliferation. The effect of brusatol on the migration and invasion of PC-9 cells was evaluated using wound healing and transwell assays, respectively. Apoptosis in lung cancer cells was detected using the Annexin V-FITC/propidium iodide assay. The correlated anticancer mechanism was detected using Western blotting. Results: The IC50 values of brusatol acting on PC-9 and H1975 cells were 1.58 ± 0.30 μM and 31.34 ± 2.72 μM, respectively, according to the MTT experiment. In addition, brusatol suppressed PC-9 cell proliferation, migration, and invasion, as well as induced apoptosis, which may be related to the downregulation of epidermal growth factor receptor (EGFR), β-catenin, Akt, and STAT3 expression. Conclusions: Brusatol showed potent anticancer activity against lung cancer PC-9 cells, inhibiting the proliferative capacity and metastatic potential of PC-9 cells. Its anticancer effect may be related to the downregulation of EGFR, β-catenin, Akt, and STAT3.
Keywords: Antitumor, brusatol, lung cancer, migration, proliferation
Traditional Chinese Medicine (TCM) is a cultural treasure with a long history that offers a new perspective for cancer treatment. In recent years, researchers seeking new drugs have focused on natural products to obtain lead compounds with evident curative effects and few side effects. Brucea javanica (L.) Merr. is a pervasively used TCM.[1] Modern pharmacological research has found that B. javanica has significant antitumor effects against diseases, such as rectal, colorectal, and esophageal cancers.[2],[3],[4] Brusatol [Figure 1], a type of nigakilactone, is a therapeutic ingredient found in B. javanica.[5] Ren et al. found that brusatol enhanced patient sensitivity to chemotherapeutics by inhibiting the cellular defense mechanism mediated by nuclear factor (erythroid-derived 2)-like 2 protein (Nrf2), which promoted the degradation of Nrf2.[6] Brusatol also induced cancer cell apoptosis by inhibiting the Akt/mTOR pathway.[5]
Figure 1: Chemical structure of brusatol and its effect on lung cancer cell viability. (a) Chemical structure of brusatol. (b) Cell viability inhibition effects of brusatol on PC-9 cells and H1975 cellsAmong all cancers, lung cancer currently has the highest fatality rate,[7] including nonsmall cell lung cancer and small cell lung cancer.[8] The former can be treated with chemotherapy, radiotherapy, and immunotherapy.[7] However, the current traditional chemotherapeutic drugs have many side effects and can easily cause drug resistance.[6] Therefore, new antitumor drugs without side effects that do not induce drug resistance are urgently needed. Recently, researchers discovered that many TCMs exhibit valuable antineoplastic effects.[9],[10]B. javanica is one such TCM, and we have been exploring the effects and mechanisms of its active ingredient, brusatol, on lung cancer cells.
Materials and MethodsCell culture conditions
The human lung cancer PC-9 and H1975 cell lines were purchased from iCell Bioscience Inc.(Shanghai, China). These cells were cultured in RPMI-1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (FBS, Gibco, CA, USA) and 1% penicillin-streptomycin solution (Guangzhou Jingxin Biotechnology Co., Ltd., Guangzhou, China) and incubated in the atmosphere of 5% CO2 and 37°C.
Reagents and antibodies
Brusatol standard was purchased from Nanjing Spring and Autumn Biological Engineering Co., Ltd.(Nanjing, China), which was diluted to 40 mg/mL with dimethyl sulfoxide (DMSO, MP Biomedicals, Inc., CA, USA) and stored at −20°C. Phosphate-buffered saline (PBS) and 0.25% trypsin were purchased from Guangzhou Jingxin Biotechnology Co., Ltd.(Guangzhou, China). MTT, TEMED, sodium dodecyl sulfate (SDS), Western Lightning® Plus-ELC kit, and ammonium persulfate were purchased from MP Biomedicals, Inc.(CA, USA). Annexin V-FITC/propidium iodide (PI) assay kit, RIPA lysis buffer, PMSF, and Tris-HCl (1.0/1.5M, pH = 6.8, 8.0, 8.8) were obtained from Beyotime Biotechnology, Inc.(Shanghai, China). Isopropanol, methanol, and ethanol were obtained from Fuyu Chemical, Inc.(Tianjin, China). BCA protein assay kit and PageRuler Prestained Protein Ladder were purchased from Thermo Fisher Technology, Inc.(Shanghai, China). Anti-epidermal growth factor receptor (EGFR) (4267S), anti-Akt (4685S), anti-STAT3 (4904S), anti-β-catenin (8480S), and rabbit anti-HRP (7074) were purchased from Cell Signaling Technology Inc.(Shanghai, China). GAPDH (MB001) and mouse anti-HRP (BS12478) were purchased from Bioworld Technology Inc.(Nanjing, China).
MTT assay
After cells were digested by trypsin, PC-9 and H1975 cells were counted under a microscope and plated in 96-well plates (3500 cells/well). The cells were incubated for 24 h. After the incubation, serial dilutions of the drug were added, and the resulting mixture was incubated for 68 h at 37°C. The 96-well plate was removed from the incubator, 5 mg/mL MTT solution (20 μL/well) was added, and the plate was incubated for 4 h. All media and MTT were removed from the plate, and 100 μL DMSO was added. The plates were then shaken. Cell viability was measured at dual wavelengths of 540 nm and 655 nm. Cell viability rate = (Aadministration group – Ablank group)/(Acontrol group – Ablank group) × 100%, where A: Absorption.[9] Administration group means the group that treated with different concentration of brusatol. Blank group means the group only added with PBS.
Colony formation assay
After harvesting the cells from the dishes, they were placed in a 12-well plate (800 cells/well) and incubated for approximately a week. When the colonies consisted of 30–40 cells, different concentrations of the drug were delivered into the wells. The drug was diluted in complete media (1, 2, and 4 μM). To better observe the colony, the medium containing brusatol was removed, and the cells were submerged in methanol (1 mL/well) for 30 min. The plates were then soaked in a 0.1% crystal violet solution for 1 h. After dying, the plates were gently rinsed with water, dried, and imaged.[10]
Wound healing assay
Three straight lines were drawn using a marker at the bottom of each 6-well plate for positioning. Cells were plated at a density of 4.8 × 105 cells/well. After the cells had attached to the bottom of the dish, a “wound” was scratched into the surface using a 200 μL sterile pipette tip. The wound was gently washed with PBS. A serum-free medium containing different doses of the drug was placed into the plate to culture the cells. Plates were photographed at three-time points: 0, 12, and 24 h. The migration ratio was computed using the following formula: Migration ratio = (1-the size of the wound at a certain time point/the size of the wound at 0 h) × 100%.[9]
Transwell assay
Matrigel and the medium without FBS were diluted at a ratio of 1:8, and 45 μL of diluted Matrigel was added to each upper chamber. The plate containing the chambers was then incubated for 2 h to allow the gel to solidify. Cells were seeded in each chamber (2.5 × 105 cells/well), and the drug solution was added at different concentrations. Next, 600 μL of a medium containing 10% FBS was added to each lower chamber for chemoattraction. Solid cells submerged in methanol were stained with 0.1% crystal violet solution after 24 h. The chambers were gently rinsed, dried, and imaged.[10]
Annexin V-FITC/PI double staining assay
To detect the impact of brusatol on cellular apoptosis, PC-9 cells (5 × 104 cells/well) were allowed to grow in a 6-well plate for 24 h and then treated with 0, 1.0, 2.0, and 4.0 μM brusatol solution. After incubation at 37°C for 48 h, the cells were dissociated, washed twice with PBS, and centrifuged. The collected cells were resuspended in Annexin V-FITC binding buffer (195 μL) and then treated with Annexin V-FITC (5 μL) and PI (10 μL), successively. Each flow tube of the mixed solution was incubated in the dark for 15 min at room temperature. The percentage of apoptotic cells was detected by flow cytometry.[10]
Western blot
Cells for the experiments were collected and washed thrice with PBS after digestion. To extract total protein, RIPA solution was added, and the cells were lysed on ice. After the protein concentration was measured, the total protein (20 μg/lane) was separated using SDS-PAGE. After electrophoresis, all proteins were transferred to a polyvinylidene fluoride membrane. The transfer conditions were set at 200 mA for 2 h. The membrane was then immersed in milk for 1 h to block nonspecific protein-binding sites. The treated membrane was subsequently submerged in a primary antibody solution (diluted antibody according to the manufacturer's instructions) and stored at 4°C overnight. The membrane was washed with Tris-HCl buffer solution with Tween-20 (TBST) after incubation and placed in a shaker at 90 r/min for 5 min × 3 times. The membrane was then submerged in secondary antibody solution in a shaker for 1 h and washed again with TBST. Chemiluminescence was used to record the membranes on the gel imager. An antibody solution of GAPDH was chosen as an internal reference, and the primary antibody solution of Akt, STAT3, EGFR, and β-catenin was used to detect protein expression.[9]
Statistical analyses
The data were expressed by x̄ ± s . Statistical analysis was performed using SPSS 25.0. Significance was considered at P < 0.05 or P < 0.01. Graphs were constructed using GraphPad Prism 8.0. A minimum of three parallel groups were designed for each experiment.
ResultsEffect of brusatol on the cell viability of PC-9 cells
As shown in [Figure 1], brusatol inhibited the viability of both the PC-9 and H1975 cells. The IC50 values for PC-9 and H1975 were 1.58 ± 0.30 μM and 31.34 ± 2.72 μM, respectively. Thus, brusatol had a clear inhibitory effect on PC-9 cells. We used PC-9 cells to conduct the following experiments.
Effect of brusatol on formation of PC-9 cell colonies
The results of the colony formation assay are shown in [Figure 2]. As the concentration of brusatol increased, the colonies became smaller and fewer in number. In summary, brusatol inhibited PC-9 cell colony formation in a concentration-dependent manner.
Figure 2: Brusatol exhibited dose-dependent inhibition against PC-9 cell colony formation. (a) PC-9 cell colony numbers after treatment with different concentrations of brusatol solution for 24 h. (b) Statistical analysis of a; *P < 0.05, **P < 0.01, and ***P < 0.001Effect of brusatol on migration and invasion of PC-9 cells
As seen in [Figure 3]a and [Figure 3]b, brusatol inhibited the migration of PC-9 cells. With the increase in drug dose, the migration distance of cells became shorter. After brusatol treatment for 12 h, the migration rates of PC-9 cells were 18.39% ± 1.60%, 9.32% ± 1.52%, 4.32% ± 2.08%, when the concentrations of brusatol were 0, 1.0, and 2.0 μM, respectively. After treatment for 24 h, the rates became 22.55% ± 1.92%, 10.93% ± 1.75%, 5.28% ± 2.78% when the concentrations were 0, 1.0, and 2.0 μM, respectively. Thus, brusatol inhibited lung cancer cell migration in a concentration- and time-dependent manner.
Figure 3: Brusatol diminished the migration and invasion ability of PC-9 cells. (a) Migration ability of PC-9 cells was inhibited by brusatol in a dose-dependent manner. (b) Statistical analysis of a; *P < 0.05, **P < 0.01, and ***P < 0.001. (c) Brusatol inhibited the invasive ability of PC-9 cells in a dose-dependent manner. (d) Statistical analysis of c; *P < 0.05, **P < 0.01, and ***P < 0.001In addition, brusatol suppressed the invasive ability of PC-9 cells. The number of PC-9 cells that passed through the pores in the chambers gradually decreased as drug concentration increased. This trend is readily observed in [Figure 3]c and [Figure 3]d.
Induction of brusatol on apoptosis of PC-9 cells
Apoptosis is programmed cell death regulated by genes and inducing apoptosis is an important mechanism used by many antitumor drugs. Annexin V, a calcium-dependent phospholipid-binding protein, can bind to phosphatidylserine with high affinity to the cell membrane. Therefore, it can be used to detect early apoptosis. PI passes through the cell membrane of midterm and late apoptotic cells and dead cells and dyes their cell cores red. These two reagents can be used to distinguish apoptotic cells at different stages. An annexin V-FITC/PI double staining assay was performed to evaluate the effect of brusatol on apoptosis in PC-9 cells. The results in [Figure 4] showed that the apoptosis percentages of PC-9 cells detected using flow cytometry were 10.08% ± 0.09%, 28.77% ± 0.57%, 39.44% ± 0.98%, and 48.46% ± 1.54% after treating the cells for 48 h with 0, 1.0, 2.0, and 4.0 μM of brusatol, respectively. In other words, brusatol-induced apoptosis in PC-9 cells in a dose-dependent manner.
Figure 4: Brusatol-induced apoptosis of PC-9 cells. (a) PC-9 cell apoptosis was detected by flow cytometry using Annexin V-FITC/PI double-staining assay after treating cells with 0, 1.0, 2.0, and 4.0 μM brusatol for 48 h. (b) Quantitative results of a. *P < 0.05, **P < 0.01, and ***P < 0.001Inhibition of brusatol on the expression of Akt, STAT3, epidermal growth factor receptor, and β-catenin
The expression of certain proteins was detected using Western blotting. The antitumor activity of brusatol may be attributed to the down regulation of the expression of Akt, STAT3, EGFR, and β-catenin, all of which can cause tumor cell proliferation, migration, and invasion, as shown in [Figure 5]. GAPDH expression was used as an internal reference, and the levels of all detected proteins were compared to it.
Figure 5: Brusatol downregulated the expressions of Akt, STAT3, EGFR, and β-catenin. (a) Expression of those proteins after treatment with different concentrations of brusatol. (b) Quantitative results of a; *P < 0.05, **P < 0.01, and ***P < 0.001. Ns: No significance Discussion Lung cancer currently has the highest fatality rate among other types of cancer.[7] Chemotherapy, radiotherapy, and immunotherapy are effective therapeutic strategies for nonsmall cell lung cancer.[8] However, traditional chemotherapeutic drugs have significant side effects, and patients are prone to develop drug resistance.[6] Therefore, new antitumor drugs are needed. In recent years, researchers have discovered that many TCMs have antitumor effects. In addition to identifying new monomers in TCMs from which antineoplastic drugs can be developed, studies have also shown that some TCMs can enhance the effect of chemotherapeutic drugs. Some TCMs can also reduce the resistance of tumor cells to antitumor drugs to a certain extent.[6] Given these discoveries, finding new antitumor drugs from TCMs has become a hot topic in antitumor research.
B. javanica is one of the most widely used TCMs.[1] Modern pharmacological studies have also found that it has antitumor effects[2] that are closely related to its chemical components, including alkaloids,[11] nigakilactone,[12] triterpenes,[13] phenylpropanoids,[14] and flavonoids.[15] Among these, nigakilactone is one of the main active components.[16] Brusatol is a type of nigakilactone found in B. javanica.
The MTT and colony formation assays indicated that brusatol, at a low dose, considerably reduced the proliferative and colony-forming abilities of lung cancer PC-9 cells. These inhibitory effects became increasingly apparent as the concentration increased. Furthermore, the results of the wound healing and transwell assays revealed that brusatol clearly prevented cancer cell migration and invasion. Tumor metastasis refers to the process of tumor cell detachment from the original lesion and spreading to remote sites to form new lesions. Cell migration and invasion are particularly important for tumor metastasis and secondary tumor formation. Finally, the Annexin V-FITC/PI double-staining assay demonstrated that brusatol could enhance apoptosis of PC-9 cells in a concentration-dependent manner.
Many biological processes, such as cell multiplication, apoptosis, and differentiation of the immune system, depend on the action of STAT3.[17],[18] Abnormal expression of STAT3 may cause tumor production, development, drug resistance, and reduced patient survival.[19] A variety of cytokines can act as ligands and bind to cell membrane receptors to phosphorylate STAT3. Phosphorylated STAT3 enters the cell nuclei and affects the expression of numerous associated proteins, such as c-Myc and survivin, regulating the cell proliferation cycle.[20],[21] At the same time, it can upregulate the expression of MMP2, MMP9, and Twist, thereby promoting tumor migration and invasion.[22],[23],[24] Increasing evidence has shown that many noncoding RNAs (ncRNAs) regulate the biological activities of cancer-related proteins, including STAT3.[25],[26],[27] For example, miR-125b-5p directly inhibits STAT3 expression.[28] The antitumor effect of brusatol may be related to this, but it needs to be proven. Akt is also involved in many biological activities;[29] it can inhibit the bioactivities of procaspase-9 and apoptosis.[30],[31] In addition, Akt can upregulate the expression of STAT3, which may have potential effects on tumor development.[32] In this study, it was discovered that one antitumor target of brusatol may be the Akt/STAT3 pathway. The drug inhibited the expression of total Akt and STAT3 proteins in lung cancer PC-9 cells, thereby inhibiting their phosphorylation to a certain extent.
Brusatol appears to inhibit not only the transduction of the Akt/STAT3 signaling pathway but also the expression of EGFR and β-catenin. EGFR is a transmembrane tyrosine kinase receptor in the erythroblastic leukemia viral oncogene homolog (ERBB) family, and its expression is closely related to tumorigenesis.[33] The gene encoding EGFR is considered to be an oncogene. It has been demonstrated that EGFR is expressed in a range of cancers,[34] and when cetuximab is used (the antibody of EGFR), the tumor lesions shrink significantly.[35] EGFR is one of the main driving forces of the downstream PI3K/Akt and MEK/ERK signaling pathways, which are largely affected by EGFR expression.[36] If these two signaling pathways are inhibited, tumor growth is inhibited to a certain extent, and tumor apoptosis is promoted.[36] Many ncRNAs, such as microRNAs and lncRNAs, influence EGFR transcription.[37],[38],[39] MiR-24 and miR-25 upregulate the expression of EGFR.[37],[38] The lncRNA UCA1 appears to inhibit the effects of EGFR-TKIs by activating the Akt/mTOR pathway.[39] This study found that brusatol inhibited the expression of EGFR in PC-9 cells in a dose-dependent manner; however, it is unknown whether it inhibits upstream ncRNAs and related downstream pathways.
β-catenin is a multifunctional protein that participates in maintaining physiological homeostasis.[40] Abnormal expression may cause many diseases, such as tumors.[40] This protein is an important component of the Wnt signaling pathway.[40] Under normal circumstances, the body controls it at a relatively low level through the ubiquitin-proteasome system.[41] When Wnt is activated or the structure of Wnt changes, β-catenin aggregates in the cytoplasm and translocates to the nucleus, where it can interact with the transcription of Jun, c-Myc, and CyclinD-1, promoting tumor formation.[42] High levels of β-catenin have been observed in various cancers, including colorectal, breast, and liver cancers.[42],[43],[44] Moreover, β-catenin has been shown to reduce immunity and promote tumor formation by inhibiting the immune response of T cells.[45] Our repeated test results showed that brusatol suppressed the expression of β-catenin in lung cancer PC-9 cells to activate its antitumor effects.
In summary, brusatol inhibited the proliferation, migration, and invasion of PC-9 cells and induced their apoptosis. Moreover, brusatol inhibited the expression of Akt, STAT3, EGFR, and β-catenin in PC-9 cells.
ConclusionsThrough a series of experiments, we found that brusatol had a considerable inhibitory effect on the proliferation, migration, and invasion of lung cancer PC-9 cells and induced apoptosis. MTT assay showed that brusatol inhibited the growth of PC-9 cells. Colony formation, wound healing, transwell, and Annexin V-FITC/PI assays revealed that brusatol dose-dependently inhibited the proliferation, migration, and invasion of PC-9 cells and induced apoptosis in PC-9 cells. The previous studies have shown that the antitumor mechanism of brusatol is related to the Akt/mTOR pathway and the expression of Nrf2. Using Western blotting, we found that the antitumor mechanism of brusatol might be related to the Akt/STAT3 pathway and the expression of EGFR and β-catenin in PC-9 cells. It is possible that phosphorylated proteins enter the nucleus to regulate the expression of related carcinogens. However, the expression of these proteins was not tested in this study. Whether brusatol can regulate the expression of downstream proteins related to tumorigenesis requires further exploration.
Financial support and sponsorship
This work was financially supported by the National Key R and D Program of China (2021YFE0202000), the National Natural Science Foundation of China (81773888), the Natural Science Foundation of Guangdong Province (2020A1515010605), and the Special Funds for the Cultivation of Guangdong College Students' Scientific and Technological Innovation (pdjh2020b0483).
Conflicts of interest
There are no conflicts of interest.
References
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