Brusatol Inhibits Laryngeal Cancer Cell Proliferation and Metastasis Via Abrogating JAK2/STAT3 Signaling Mediated Epithelial-Mesenchymal Transition

AIMS
This study aimed at investigating the role of Brusatol (BR) on human laryngeal squamous carcinoma cell (Hep-2) to study its underlying mechanism through in vitro and in vivo approaches.


MATERIALS AND METHOD
In the present research, we employed various cell-based assays, such as cell proliferation, apoptosis, cell cycle assessment, migration and invasion assays were used to examine the anti-tumor effect of BR on Hep-2 cells. Immunohistochemistry (IHC), qRT-PCR and Western blotting were performed to study the underlying molecular mechanisms. To validate our in vitro findings we used a subcutaneous tumor-bearing model of Balb/c mice with Hep-2 cells of laryngeal carcinoma (LC) to study the inhibitory effect of BR on Hep-2 cells in vivo.


KEY FINDINGS
The results indicated that BR markedly inhibited the viability, migration and invasion capacity of Hep-2 cells, with no significant toxic effect on normal Human bronchial epithelial cell line (BEAS-2B). Also, BR induced cellular apoptosis by blocking the cells in S phase to suppress cell proliferation. Immunohistochemistry results revealed that BR inhibited the protein expression levels of epithelial-mesenchymal transition (EMT)-related markers. Mechanistically, western blotting results exhibited that BR could suppress the protein expression of both JAK2/STAT3 and their phosphorylation levels. Our in vivo experiments further validated the anti-tumor effect of BR on Hep-2 cells in vitro, where BR suppressed the growth of xenograft laryngeal tumor without apparent toxicity.


SIGNIFICANCE
The present study highlights the anti-LC effect of BR by possibly abrogating JAK2/STAT3 signaling mediated EMT process. BR may be a promising therapeutic candidate for the treatment of LC.


Introduction
Laryngeal cancer (LC) is the second most widespread malignancy of the upper aerodigestive tract squamous cell carcinoma, which is characterized by hoarseness, sore throat and persistent cough [1,2].About 95% of laryngeal carcinoma are diagnosed as laryngeal squamous cell carcinoma (LSCC) [3].In recent decades, due to excessive tobacco and alcohol consumption, the incidence rate of laryngeal carcinoma has been gradually increased [4].Currently, surgery and chemotherapy are the most common therapies for the treatment of LC [5].Nevertheless, chemotherapeutic drugs are always resulted in incomplete clinical e cacy and serious side effects, and surgery frequently lead to the loss of language function [6,7].Therefore, the development of novel and secure therapeutic alternatives is urgently needed.Recently, anti-tumor agent from natural plants is one of alternative treatment options and has been increasingly recognized worldwide.
Brucea javanica, a plant distributed widely throughout Asia, especially in Southern China, has been used in Traditional Chinese Medicine for treating various diseases [8].The quassinoids are the main compounds of Brucea javanica, and largely responsible for its diverse bioactivity [9].Brusatol (BR, the structure shown in Fig. 1A), an important quassinoid of Brucea javanica, has been rapidly recognized as a potent anticancer agent in the past few decades [10].The key mechanism for the anticancer activity of BR is suppression of protein synthesis.Importantly, Recent researches have revealed that diverse protein targets of BR were involved in its anticancer activity [11].Such as, it has been proved to sensitize numerous cancer cells in vitro and A549 mouse xenografts by uniquely blocking the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway [12].Intriguingly, STAT3 was postulated to be an effector molecule downstream of Nrf2 [13].As is well-known, JAK2/STAT3 is a crucial signaling pathway involved in cell proliferation, apoptosis, angiogenesis, migration, invasion and metastasis, and has a signi cant in uence on the occurrence and development of tumors [14].Moreover, it is well known that this signaling pathway regulate epithelial cell-cell adhesion and is intimately associated with the occurrence of EMT in malignant lesions [15].Currently, it has been discovered that EMT played a key role in the early tumor metastasis, which can render tumor cell with migration and invasion capacities as well as stem cell characteristics [16].Meanwhile, previous studied has demonstrated that BR could effectively inhibit tumor cell invasion and sensitize the current rst-line chemotherapeutic agents in hepatocellular carcinoma and pancreatic cancer via inhibition of the EMT process [17,18].
Based on the above considerations, our present study is to investigate the potential inhibitory effects of BR in human laryngeal carcinoma Hep-2 cell line from the perspective of EMT process and JAK2/STAT3 signaling pathway, aiming to clarify its underlying molecular mechanism into the anti-LSCC effect of BR.

Cell lines and culture
Human laryngeal squamous carcinoma cell line Hep-2, human esophageal cancer cell line K150 and human ovarian cancer cell line SK-OV-3 were purchased from the China Center for Type Culture Collection and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS).Human bronchial epithelial cell line BEAS-2B, human hepatoma cell line HepG2, human non-small cell lung cancer cell line A549 and human colorectal cancer cell line Caco-2 (EK-Bioscience, Shanghai) were cultured in DMEM supplemented with 10% heat-intactived FBS, 1% streptomycin/penicillin in a humidi ed atmosphere with 95% air and 5 % CO 2 at 37 ℃.

Morphological observation
Hoechst 33258 staining assay (Beyotime Biotechnology, Shanghai, China) was applied to observe the morphological characteristics of apoptosis cells.Shortly, the Hep-2 cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well and incubated with BR (0, 0.2, 0.4, 0.6 µM) for 24 h.Then, cells were xed with 4% polyoxymethylene and washed three times with PBS, followed by incubated for 15 mins in the dark with Hoechst 33258 (10 µg/mL) and washed three times with PBS.The morphology of apoptotic cells was observed under the uorescence microscopy (DMi8, Leica, Germany).The experiment was repeated three times.

Cell cycle assay
Cells were inoculated into 6-well plates at a density of 1 × 10 6 cells/well and treated with different concentration of BR (0, 0.2, 0.4, 0.6 µM) for 24 h.For cell cycle assay, cells were washed with PBS, harvested by trypsinization and xed with 70% ethanol at 4 ℃ for 5 h, then stained with Propidium Iodide (PI, Keygen Biotech, China) in dark at 37°C for 30 mins.Determination of stained cells was by using FCM (BD Bioscience, USA).The experiment was repeated three times.

Cell apoptosis assay
Apoptosis was analyzed with the Annexin V-FITC Apoptosis Detection Kit (Keygen Biotech, China) following manufacturer's instructions.Cells were exposed to different BR (0, 0.2, 0.4, 0.6 µM) for 24 h.The treatment cells were harvested with EDTA-free trypsin and washed with cold PBS, then the cells were resuspended in 200 µL binding buffer and stained with 5 µL Annexin V-FITC and 5 µL Propidium Iodide in dark for 30 mins.Determination of stained cells was by using FCM within 1 h.Data were analyzed by FlowJo software (Tree Star, OR, USA).The experiment was repeated three times.

Transwell assay
Transwell assay was applied to evaluate the Hep-2 cells migration and invasion.Brie y, the treatment cells were resuspended with 4 × 10 4 cells in 200 µL serum-free RPMI-1640 and added into the upper chambers of Matrigel-uncoated and -coated transwell inserts (24 plate with 8 µm pore diameter, Corning, Inc., Corning, NY, USA).The lower chamber was lled with 200 µL RPMI-1640 containing 20% FBS at 37 ℃ for 48 h, the cells that had migrated or invaded to the lower chamber were xed with 4%paraformaldehyde in PBS buffer and stained with crystal violet (0.1%).The cells were observed in a microscope and cells from at least three randomly selected elds were counted.Each concentration was repeated 3 chambers and cells from at least 5 randomly selected elds were counted.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from the prepared cells using TransZol UP reagent (Transgen, Beijing, China), and 1 µg of total RNA was reversely transcribed into cDNA using one-step gDNA Removal and cDNA Synthesis Supermix kit (Transgen, Beijing, China) according to the manufacturer's instructions.Next, qRT-PCR analyses using Tip green qPCR Supermix in the Light Cycler 96 Real Time PCR System (Roche, Mannheim, Germany).The used primers were purchased from Sangon Biotech Co. Ltd. (Shanghai, China).Table 1 has listed the sequences of primers.Thermal cycling conditions were as follows: 30 s at 94 ℃, 40 cycles of 5 s at 94 ℃ and 30 s at 60 ℃.The fold-change of expression was calculated using the 2 −ΔΔCt method and normalized by 18s.

Table 1
Sequences of the primers used in the real-time PCR

Target
Forward primer sequence (5'-3') Reverse primer sequence (5'-3') Immunohistochemistry Analysis (IHC) Immunohistochemistry was performed according to our previous method [20].Brie y, the para nembedded cell slices were dipped into xylene and graded ethanol in order to dewax and rehydrate.After antigen retrieval and endogenous peroxidase activity is blocked, the sections were incubated overnight with rabbit Vimentin, E-cadherin, N-cadherin and Snail polyclonal antibody (Abcam, USA) at 4°C, respectively.After washed three times, the slices were successively subjected to the rest of the process, including incubation with biotin-linked secondary antibody, PBS washing, streptavidin-HRP incubation, DAB staining and hematoxylin counterstaining.Finally, the pictures were captured with the microscope imaging system microscope.The integrated optical density (IOD) and of each photograph was analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockvile, MD, USA).

Western Blot Analysis
Cell samples were lysed on ice in RIPA buffer (Servicebio, Hubei, China) containing 1% PMSF and 1% phosphorylated inhibitors.The BCA protein assay kit was used to determine the protein concentration and after separation on 10% SDS-polyacrylamide gel electrophoresis, 20 µg of protein was electrically imprinted on PVDF membrane.The immunoblots were then incubated with antibodies to STAT3, p-STAT3, JAK2, p-JAK2 and β-actin (Abcam, USA) respectively.All intensities of the target protein bands were detected using ChemiDocRS imaging systems and analyzed using Image lab software 6.0 (Bio-Rad Laboratories).
Animal Model

HE staining
The tissue sections were dewaxed, then dyed with hematoxylin and Eosin (H&E), rinsed with distilled water, dehydrated with gradient anhydrous ethanol, transparently treated with xylene, and then sealed with neutral gum.The sections were observed and photographed under a Nikon ECLIPSE E100 optical microscope (Nikon, Tokyo, Japan).

Statistical Analysis
All experiments were repeated at least three times.Statistical analyses were performed using SPSS software (version 26.0, IBM, New York, USA) and GraphPad Prism version 6.0 (GraphPad software, San Diego, CA, USA).All values were presented as mean ± SD.One-way ANOVA analysis was performed for comparison of multiple means.Statistical signi cance was set at P < 0.05.

Results
The inhibition effects of BR on proliferation of cancer cells Human cancer cell lines of six different histotypes were used to investigate the anti-proliferation activities of BR by Cell Counting Kit-8 (CCK-8) assay for 24 h, and the results indicated that the inhibitory effect was obviously different between different cancer cell lines.Intriguingly, we found that the human laryngeal carcinoma cell line Hep-2 was the most sensitive to the treatment of BR, with an IC50 value of about 0.53 µM, followed by Hep-2(0.55µM),K150 (0.70 µM), Caco-2 (0.70 µM), A549 (1.91 µM), SK-OV-3 (1.61 µM) and HepG2 (1.28 µM)) (Fig. 1B).
The selectivity of BR was assessed using normal cell lines After incubated with designed doses for 24 h, 48 h and 72 h, the cytotoxicity of BR was detected in the human laryngeal cancer cell line Hep-2 and the normal human bronchial epithelial cell line BEAS-2B.The results demonstrated that BR markedly inhibited the proliferation of the Hep-2 cells in a time and concentration-dependent manner (Fig. 1C).The calculated IC50 values at 24, 48 and 72 h were (0.53 ± 0.13, 0.44 ± 0.08 and 0.37 ± 0.10 µM), respectively.Nevertheless, no signi cant difference of cell viability was observed in a normal bronchial cell line BEAS-2B after treated with BR for 24, 48 and 72 h (Fig. 1D).

BR induces cell apoptosis in Hep-2 cells
To investigate whether BR-mediated cytotoxic effect on Hep-2 cells was related to the induction of apoptosis or necrosis, Hoechst 33258 nuclear staining was performed.As shown in Fig. 2A&B, compared with the control cells, the cells treated with BR exhibited a signi cantly increase in the levels of apoptotic chromatin condensation, nuclear fragmentation and shrinkage as well as the number of dead cells.Data from Flow cytometry analysis (FCM) also demonstrated that BR treatment generated more apoptotic cells (2.65 ± 0.48%, 11.58 ± 2.56%, 25.95 ± 1.89% at 0.2, 0.4 and 0.6 µM of BR, respectively) than that in the control group (1.52 ± 1.10 %) (Fig. 2C and 2D).

BR elicits S cell cycle arrest in Hep-2 cells
In order to further determine whether the anti-proliferation activity of BR in Hep-2 cells is related to cell cycle arrest, Hep-2 cells were respectively exposed to different concentrations of BR (0, 0.2, 0.4, 0.6 µM) for 48 h, and FCM was employed to analyze the cell cycle distribution.As shown in Fig. 2E and 2F, compared with the control cells, BR treatment resulted in a marked accumulation of S-phase cells, and reduced the percentage of cells in G1 phase.Quanti cation of cell cycle distribution suggested that BR treatment caused the cell cycle to stagnate in S phase and induced cell apoptosis in Hep-2 cells in a dose -related fashion.

BR suppresses the metastasis of Hep-2 cells
Then, we evaluated the effects of BR on cell migration and invasion using transwell assay.As indicated in Fig. 3, migration and invasion of Hep-2 cells were signi cantly inhibited after BR exposure in a concentration-dependent manner, meanwhile the untreated cells were found to actively invade through the matrigel.

BR reverses the EMT process of Hep-2 cells
EMT is an important physiological process in the migration and invasion of malignant tumor cells [21].
Following treatment with 0.2, 0.4 and 0.6 µM BR for 48 h, IHC analysis was performed to evaluate the EMT-associated proteins.Compared with the control group, BR treatment groups decreased the expression of Hep-2 cells mesenchymal markers N-cadherin, Vimentin, snail and upregulated the expression of the epithelial marker E-cadherin (Fig. 5), the regulation of EMT markers appeared to be dependent on the dose of BR applied.Additionally, in parallel with the IHC experimental results, the above results were also veri ed by qRT-PCR analysis (Fig. 4A, 4B, 4C and 4D).As expected, the mRNA expression of these EMT-related markers was regulated by BR in a dose-dependent manner.These observations suggested that BR diminished the EMT process in Hep-2 cells.

BR abrogates the JAK2/STAT3 signaling pathways in Hep-2 cells
To further elucidate the potential mechanisms of the anti-tumor activity exerted against Hep-2 cells by BR, the expression levels of JAK2/STAT3 and their phosphorylated forms after exposure to BR were investigated.As shown in Fig. 6, compared with the control group, BR dose-dependently suppressed the protein expression levels of JAK2/STAT3 as well as phosphorylation levels in Hep-2 cells.In addition, qRT-PCR analyses were also carried out to clarify whether the reduction of protein levels of STAT3 and JAK2 were due to the inhibition of gene expressions.As exhibited in Fig. 4E and 4F, the mRNA levels of STAT3 and JAK2 were down regulated by BR in a dose-dependent manner.All above results indicated that BR not only inhibited the phosphorylation of JAK2/STAT3 but also negatively regulated the gene expression of JAK2/STAT3 in Hep-2 cells.In summary, these data suggested that BR could abrogate the activity of JAK/STAT3 signaling pathway in Hep-2 cells.

BR Inhibited Tumor Growth in the Hep-2 Subcutaneous Tumor Xenograft Mouse Model
Male Balb/c mice with Hep-2 tumor cells were used to evaluate the anti-tumor activity of BR in vivo.The experimental protocol is depicted in Fig. 7A.The anti-tumor activities were assessed by comparing body weight, tumor volume, tumor weight and changes in various organs and tissues.The morphological changes of the tumor were illustrated in Fig. 7G, and the growth of laryngeal cancer was obviously inhibited by BR group and Cis group.After 20 days administration, BR at doses of 1.0 and 2.0 mg/kg and Cis at dose of 5 mg/kg signi cantly decreased in the tumor weights by 42.15 ± 8.29%, 57.55 ± 4.91% and 53.09 ± 4.85%, respectively (Fig. 7D).The tumor volumes were increased slower in the 1.0 and 2.0 mg/kg BR and Cis groups than in the control group (P < 0.05) (Fig. 7B).It was worth noting that mean body weight was not signi cantly different in BR and control group (P > 0.05).Whereas the weight of Cis group signi cantly lower than the control group (P < 0.05), suggesting that compared to Cis group, BR exhibited fewer side effects (Fig. 7C).By calculating spleen and thymus index, it was found that compared with the positive drug (Fig. 7E&F), BR group could maintain the spleen and thymus quality of mice at normal level, indicating that the side effects of BR were not obvious, and its tumor inhibition mechanism might be different from that of cisplatin.
To further explore the toxicity of the drug in vivo.The heart, liver, spleen, lung, kidney and tumor tissues were made into sections and H&E stained to evaluate the toxicity of BR on different tissues.The results were shown in Fig. 7H.The kidneys, lungs, spleen, liver and heart in the normal group (normally fed mice without tumor inoculation), the control group and the BR group showed no damage.However, the Cis group caused a small amount of hepatocyte necrosis at the edge of the liver tissue of the mouse, and the nucleus was deeply stained and fragmented, and the cytoplasm was increased in eosinophilia (red arrow), which caused liver damage.A large amount of renal tubular epithelial edema at the junction of the cortex and medulla of the kidney tissue, swelling of the cells and faintly stained cytoplasm (black arrow) cause kidney damage.
At the same time, we observed that the tumor cells in the control group were tightly arranged and disordered, with larger nuclei, rich cytoplasm, darker staining and less stroma.A large amount of in ammatory cell in ltration can be seen at the edge of tumor tissue in BR group.And we can observe that nuclei were abnormal, high nucleo-plasm ratio, nucleolus was not obvious and mitotic phase was rare.Furthermore, it's still observable that moderate cell necrosis, nucleus pyknosis, fragmentation or lysis eosinophilia is enhanced, forming a typical phenomenon of apoptotic bodies.Taken together, these results strongly suggested that BR inhibited the growth of Laryngeal cancer in vivo.

Discussion
LSCC has the second highest incidence among Head and neck squamous cell carcinoma (HNSCC) and is especially prevalent in the northern areas of China, including Shanxi Province [22].Due to adverse effects and poor prognosis of the existing treatments, it is extremely urgent to explore alternative anticancer drugs with appreciable e cacy and minimized toxic effects from medicinal plants.BR is the primary active component of Brucea javanica, and has been reported to possess effective extensive bioactivities including anti-in ammatory, antitumor, antimalarial, antiviral and insecticide activities [20].In spite of various works have evaluated the vital roles of BR in tumors, reports on the role of BR in LSCC are rare.
Our work also proved that BR exerted potent growth inhibitory activities against a broad spectrum of human cancer cell lines.Among these cells, Hep-2 cells were the most sensitive to BR.Hence, in the current study, we rstly use a human laryngeal carcinoma Hep-2 cell line to investigate the effect of BR on LSCC and explored its mechanism of action.
The proliferative capacity of tumor cells is considered pivotal for the growth and development of tumors [23].In the current research, we rstly exposed Hep-2 cell line to increasing concentrations of BR at different time periods and the IC50 values were evaluated using a CCK-8 method.The results demonstrated BR signi cantly inhibited the proliferation of Hep-2 in a dose-and time-dependent manner.Meanwhile, mild cytotoxic effect was observed on normal human bronchial epithelial cell line (BEAS-2B), indicating that BR might be a highly selective killing agent toward laryngeal cancer cells in a certain concentration range.
Apoptosis is a programmed cell death process which includes series of biochemical events leading to several vital featured morphological alternations, for example, cell shrinkage, chromatin condensation, and nucleus DNA fragmentation [24].The induction of apoptosis is presumed to factor prominently in most anti-tumor therapy mechanisms [25].The results displayed indicated that the cell morphology changed obviously: the cell edge was abnormal, the adherence ability declined, the chromatin condensed, the uorescence became dense, and bright blue spots (apoptotic bodies) could be seen.In parallel with the morphological results, Annexin V-FITC/PI double staining also con rmed that BR could induce Hep-2 cells apoptosis.
Cell cycle is the essential mechanism during reproduction of all living things [26].Loss of cell cycle control is one of the typical characteristics of tumorigenesis [27].In this study, we have found that BR induction markedly increased proportion of S phase in Hep-2 cells.These results suggested that BR might result in cell cycle arrest and result in DNA fragmentation.
Tumor metastasis is a complicated course which acts as a considerable malignant behavior in the process of tumor development and progression.Migration and invasion are two different cellular forms in tumor metastasis [14].In our study, different concentrations of BR were used for transwell assays, which indicated that BR signi cantly inhibited Hep-2 cells migration and invasion in a dose-dependent manner, suggesting that BR exerts a potent anti-migratory effect on Hep-2 cells.EMT, one of the distinctive characteristics of tumor metastasis, is a biological process featured by cellular and molecular changes [28].When EMT occurs, the expression of an epithelial marker, such as E-cadherin, is reduced, whereas that of mesenchymal markers, such as N-cadherin, Vimentin and Snail, are increased [29].Hence, a reversal of the EMT process is regarded as a latent strategy to ameliorate the migration and invasiveness of malignant tumors.Immunohistochemistry analysis and qRT-PCR results revealed that BR inhibited the activation of EMT via the downregulation of mesenchymal biomarkers N-cadherin, Vimentin, Snail and upregulation of the epithelial biomarker E-cadherin, consequently suppressed migration and invasion in cancer cells.These ndings imply that the BR may possess the ability to control metastasis of laryngeal cancer.
STAT3 is a potential transcription factor, it acted as a pivotal role in the transcriptional activation of cell apoptosis and cycle progression [30].STAT3 can be activated by the activation of JAK2, a non-receptor tyrosine kinase.Upon activation, STAT3 undergoes phosphorylation-induced homodimerization and translocates into the nucleus where it regulates its target gene [31,32].The JAK2/STAT3 signaling pathway is constitutively activated in varieties of malignant tumors and plays a critical role in multiple biological processes [33].Thus, inhibition of this signaling pathway is considered as a promising therapeutic target for cancer therapy.Moreover, a series of studies have proved that the activation of JAK2/STAT3 pathway devotes to progression of EMT in multiple cancer cells [34].Previous reports indicated that the phosphorylation of STAT3 is overactive in Hep-2 and TU212 cells and implicated in cell proliferation, invasion and migration [35].In our current study, the western blotting results revealed that BR could inhibit the JAK2/STAT3 signaling pathway by reducing the protein expression and phosphorylation levels in Hep-2 cells.
In addition, the anti-tumor effect of BR was further veri ed by in vivo experiment.At a dose of 2 mg/kg, BR signi cantly inhibited tumor growth in a xenograft model of laryngeal cancer, the tumor volume was reduced by about 30%, which was comparable to the Cis group.Moreover, the Cis group caused a signi cant weight loss (16%) in mice, while there was no signi cant difference in weight between the BR group and the control group.H&E staining demonstrated that cisplatin caused liver and kidney damage, and BR has no effect on vital organs.Therefore, our in vivo experimental results provide additional support for BR as an effective and non-toxic treatment for LC.
In summary, our study is the rst one to de ne the mechanism of activity of BR in human laryngeal carcinoma.These novel ndings demonstrated that BR was found to suppress the tumor growth both in vivo and in vitro, at least partially, through its abrogation of JAK2/STAT3 signaling pathway mediated-EMT process.Our pioneering endeavor also manifested that BR exerted similar anti-laryngeal cancer effect as the current rst-line agent cisplatin with much smaller dosage and advantageous safety pro le.All these ndings provide solid evidence that BR may be an attractive candidate drug for future laryngocarcinoma therapeutics.Moreover, all these effects need to be investigated using clinical samples and further studies are underway.