JSI‐124 inhibits cell proliferation and tumor growth by inducing autophagy and apoptosis in murine malignant mesothelioma

Malignant pleural mesothelioma (MPM), mainly caused by asbestos exposure, has a poor prognosis and lacks effective treatment compared with other cancer types. The intracellular transcription factor signal transducer and activator of transcription 3 (STAT3) is overexpressed and hyperactivated in most human cancers. In this study, the role of STAT3 in murine MPM was examined. Inhibition of the Janus kinase 2 (JAK2)/STAT3 pathway with the selective inhibitor JSI‐124 has an antitumor effect in murine MPM. Specifically, we demonstrated that JSI‐124 inhibits murine MPM cell growth and induces apoptotic and autophagic cell death. Exposure of RN5 and AB12 cells to JSI‐124 resulted in apoptosis via the Bcl‐2 family of proteins. JSI‐124 triggered autophagosome formation, accumulation, and conversion of LC3I to LC3II. Autophagy inhibitors, Chloroquine (CQ) and Bafilomycin A1 (Baf‐A1), inhibited autophagy and sensitized RN5 and AB12 cells to JSI‐124‐induced apoptosis. Our data indicate that JSI‐124 is a promising therapeutic agent for MPM treatment.


| INTRODUCTION
Malignant pleural mesothelioma (MPM) is a rare and highly lethal cancer that usually develops in the surface layer of the lungs, known as the pleura.The main risk factor for MPM is asbestos exposure under occupational conditions 1 ; however, an association between nonoccupational exposure to asbestos and MPM has also been noted. 2,3Recently, multi-walled carbon nanotubes (MWCNT-7) have been reported to induce malignant mesothelioma in experimental animals. 3A prolonged latency period between the first asbestos exposure and the development of MPM has been established.
Owing to the aggressive and refractory characteristics of MPM, its early diagnosis and treatment are critical.However, diagnosis and treatment remain challenging for clinicians.Although the benefits of trimodality therapy for MPM have not yet been defined, it is considered the best treatment strategy. 4,5According to the guidelines of the National Comprehensive Cancer Network, the preferred regimen of first-line systemic chemotherapy includes a combination of pemetrexed and platinum. 6Bevacizumab, nivolumab, and ipilimumab are also included under the principles of systemic therapy under certain circumstances. 7,8though some clinical investigations have shown promising results, second-line treatments for MPM are lacking.Therefore, finding novel drugs for MPM treatment is necessary.JSI-124, also known as cucurbitacin-I (Cu-I), is a chemical compound belonging to the cucurbitacin family and has anticancer properties.It was originally identified as a potent selective inhibitor of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in multiple cancer cell lines. 91][12] JSI-124 also inhibited cell proliferation and cell-cycle arrest. 13Additionally, JSI-124 reduces radioresistance in head and neck squamous carcinoma. 14However, the exact mechanism underlying STAT3 inhibition by JSI-124 in MPM remains unclear.
This study aimed to investigate the antitumor effects of JSI-124, a STAT3 inhibitor, and identify the downstream pathways and target genes affected.

| Cell lines and cultures
The human lung adenocarcinoma cell line (A549) was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China).A549 was cultured in RPMI 1640 supplemented by 10% fetal bovine serum and 100 μg/mL penicillinstreptomycin in 5% CO 2 in a humidified incubator at 37°C.The murine mesothelioma cell line RN5, derived from C57BL/6 mice, was recently established by Blum et al. 15 and the cell line AB12 (from Balb/c mice) was provided by Dr. Jay Kolls, University of Pittsburgh (Pittsburgh, PA).Both cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin and maintained at 37°C in an atmosphere containing 5% CO 2 . 16The cell lines were generously provided by Dr. Marc de Perrot Laboratory.
The cell lines were regularly tested and maintained negative for Mycoplasma spp.

| Cell viability assay
Cells were seeded in a 96-well plate at a density of 8000 cells per well.After 24 h, the cells were treated with JSI-124 at various doses for 48 h or 72 h.Inhibitory effects were determined using the sulforhodamine B (SRB) assay. 17The optical density was measured at 510 nm using a microplate reader (TECAN).The percentage cell viability was calculated using the formula: ODsample/ODcontrol.The half-maximal inhibitory concentration (IC50) values were calculated using Prism-GraphPad software.

| Apoptosis analysis
Cells were harvested, resuspended in a binding buffer, and incubated with annexin V-propidium iodide (AV-PI) double staining was performed according to the manufacturer's protocol.Apoptotic cells were detected by flow cytometry (BD Biosciences, AccuriTM C6), and the results were analyzed using FlowJo software (LLC).Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat No. 74106) according to the manufacturer's instructions, and the quality was verified by 1% agarose gel electrophoresis.
Agilent Bioanalyzer 2100 (Agilent Technologies) and Nanodrop-2000 spectrophotometer (Thermo Scientific) were used to evaluate the quality and purity of the RNA samples based on an RNA integrity number (RIN) of ≥8.0.mRNA was isolated from 5 μg of total RNA that had been extracted using Dynabeads Oligo (dT) (Thermo Fisher) and purified twice.Fragmented RNA molecules were used to synthesize complementary DNA (cDNA) using SuperScript II Reverse Transcriptase (Invitrogen, cat.1896649).The resulting cDNA was subjected to second-strand synthesis using Escherichia coli DNA polymerase I (NEB, cat.m0209),RNase H (NEB, cat.m0297), and dUTP Solution (Thermo Fisher, cat.R0133), resulting in the synthesis of U-labeled second-stranded DNAs.An A-base was added to the blunt ends of each strand to prepare the U-labeled secondstranded DNA for ligation to the indexed adapters.The indexed adapters used for ligation of the A-tailed fragmented DNA had a T-base overhang, which allowed them to be ligated to the A-tailed ends of the DNA.Following the ligation of dual-index adapters to the DNA fragments, size selection was performed using AMPureXP beads.After treatment with the heat-labile UDG enzyme (NEB, cat.m0280), which removed the uracil residues from the U-labeled second-stranded DNAs, the ligated products were amplified using polymerase chain reaction (PCR).
The average insert size of the final cDNA library was 300 ± 50 bp.

| Sequence and filtering of clean reads
The reads obtained from sequencing machines consist of raw reads that may contain low-quality bases or adapters, potentially affecting downstream assembly and analysis.To obtain high-quality clean reads, we employed Cutadapt (https://cutadapt.readthedocs.io/en/stable/, version: cutadapt 1.9) to filter the reads further.The following parameters were used for the filtration process: removal of reads containing adapters reads containing polyA and polyG, reads containing more than 5% unknown nucleotides (N), and low-quality reads containing more than 20% low-quality (Q-value ≤ 20) bases.To ensure sequence quality, we employed FastQC (version 0.11.9) to evaluate the Q20, Q30, and GC content of the clean data.The assessment of these metrics is crucial for determining the accuracy and reliability of the sequencing data.
The p values obtained were subjected to adjustment using the Benjamini and Hochberg method to control the false discovery rate (FDR).Genes showing an adjusted p value (Padj) < 0.05 (FDR < 0.05), as determined by DESeq.2, and with a fold-change (FC) of >2 between two groups were considered as differentially expressed.
DEGs in three comparisons of JSI-124 750 nM treatment group versus DMSO group, JSI-124 500 nM treatment group versus DMSO group, and JSI-124 750 nM treatment group versus 500 nM treatment group are listed in Supporting Information: Table S1.

| Gene set enrichment analysis (GSEA) and mfuzz analysis
GSEA (Version 4.3.2) was performed to analyze the differences between the two groups: the DMSO group versus the 750 nM group and the 500 nM group versus the 750 nM group.The statistical settings for GSEA were as follows (number of permutations = 1000, max size: exclude larger sets = 500, min size: exclude smaller sets = 15).Gene sets in the signaling pathways with |NES| > 1, NOM p-value < 0.05, and FDR q-val<0.25 are considered statistically significant.
Mfuzz analysis was performed using default parameters to divide all the genes obtained by RNA-seq into 12 clusters. 19,20A total of 960 genes included in Cluster 11 and 1091 genes included in Cluster 12 were combined for further Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.

| Cell-cycle analysis
RN5 cells were treated with various concentrations of JSI-124 for 24 h or 48 h.After treatment, cells were harvested and washed twice with cold phosphate-buffered saline (PBS).The cells were then fixed and permeabilized with 70% ethanol at −20°C for at least 30 min.
After washing, the fixed cells were incubated at 37°C in PBS containing RNAase A for 30 min, subsequently exposed to DNAinteracting dye propidium iodide (PI) (10 μg/mL) at 37°C for half an hour in the dark.Data were acquired using a flow cytometer (AccuriTM C6; BD Biosciences).

| Western blot analysis
The preparation of whole-cell protein lysates and western blot analysis procedures have been previously described. 21Briefly, cell lysates were prepared in radioImmunoprecipitation assay lysis buffer containing protease (Sigma-Aldrich, Merck KGaA) and phosphatase inhibitors (Roche Applied Sciences).The total protein concentration was determined using the Pierce™ bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.).A total of 20 μg whole-cell protein was separated by 10% or 12.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and was electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes.After completing protein transfer, the PVDF membrane was stained with Ponceau S solution to observe the integrity and consistency of the protein bands across all lanes.Subsequently, the membranes were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies.
The numbers indicated below the blots represent the relative quantification determined by densitometry by normalizing the DMSO group levels, with the reference point set to 1. Data from three separate experiments were collected for analysis.

| Transmission electron microscopy (TEM)
Cells were fixed with 3% glutaraldehyde in PBS and postfixed with 1% OsO 4 in 0.1 M cacodylate buffer for 2 h at 4°C.After staining with 1% Millipore-filtered uranyl acetate, the samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in an epoxy resin (ZXBR, Spon 812).Electron photomicrographs of the RN5 cell ultrastructures were obtained using a TEM (JEM-1200EX II, JEOL).

| Virus production
The plasmids red fluorescent protein-green fluorescent protein-LC3 (RFP-GFP-LC3b (Hedgehogbio), psPAX2 (#12260, Addgene), and pMD2.G (#12259, Addgene) were cotransfected at a ratio of 1:1:1 into HEK293T cells using the Lipofectamine 3000 reagent (Invitrogen Life Technologies) according to the manufacturer's instructions.HEK 293T is a derivative of the original HEK293 parent cell line, which is commonly used for retroviral production.Lentivirus-containing supernatants were harvested 48 and 72 h later.The supernatants were collected and filtered through a 0.45-μm filter and viruses were collected by ultracentrifugation at 110,000g for 120 min.The viral particles were then diluted and used to infect RN5 and AB12 cells.

| Cell transfection and RFP-GFP-LC3 dot assay
RN5 and AB12 cells were seeded into 12-well plates at a density of 1 × 10 4 cells/well 24 h before transduction.The cells were then incubated with lentivirus.Forty-eight hours after transduction, the medium was replaced with fresh medium containing 6 μg/mL puromycin.Accordingly, a lentiviral infection was performed.Cells displaying ≥3 GFP-LC3b dots after JSI-124 treatment were considered autophagic and were counted.The images were acquired using an LSM 880 Confocal Microscope (Carl Zeiss Microscopy).

| TUNEL staining
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed using In Situ apoptotic cells detection kit following the manufacturer's protocol (Promega, G7130).Sections were evaluated under an Olympus BX43 light microscope (Olympus Corporation).TUNEL-positive cells were counted in at least 100 random fields using a light microscope (Olympus BX43).

| Statistical analysis
Statistical analysis was performed using GraphPad software.Data are expressed as mean ± S.D from at least three separate experiments.
Differences between groups were analyzed using a one-way analysis of variance followed by the least significant difference.Statistical significance was set at p < 0.05 level.

| JSI-124 inhibits cell proliferation in murine MPM cells dependently on its anti-STAT3 activity
To determine whether JSI-124 was effective against murine MPM, RN5, and AB12 cells were treated first with different doses of JSI-124 in vitro using the SRB cell viability assay.RN5 and AB12 cells were treated with 250, 500, and 750 nM JSI-124 for 48 h, while a ZHANG ET AL.
| 1891 serial dose of JSI-124 was incubated with RN5 and AB12 cells for 72 h resulting in IC50 values (Figures 1A,B).The results showed that cell viability decreased with the dose at 48 h.The 72 h IC50 of JSI-124 was 0.599 μM in RN5 cells and 1.375 μM in AB12 cells (Figure 1B), while the IC50 of cisplatin was 7.19 μM in RN5 cells, which was reported in our previous study. 21The IC50 of cisplatin was 1.428 μM in AB12 cells (Supporting Information: Figure S1A).These results indicate that JSI-124 inhibits cell proliferation in a dosedependent manner, whereas JSI-124 exhibits higher cytotoxicity compared with cisplatin in inhibiting cell proliferation.
According to a previous study, A549 cells express high levels of tyrosine (Y705)-phosphorylated STAT3 (p-STAT3). 9To determine whether RN5 and AB12 cell lines also exhibit high levels of p-STAT3, A549 cells (lung adenocarcinoma) were selected as positive controls for p-STAT3.Among the three cell lines, RN5 and AB12 cells showed high levels of p-STAT3 (Supporting Information: Figure S1B).RN5 and AB12 cells were treated with 250, 500, or 750 nM JSI-124 for 48 h.JSI-124 at concentrations of 250, 500, and 750 nM reduced the levels of p-STAT3 in RN5 and AB12 cells (Figure 1C).The levels of p-JAK2 did not change after treatment with JSI-124.These results indicate that STAT3 may play a critical role in cell proliferation.

| Transcriptome alterations of murine RN5 cells treated by JSI-124
To investigate the role of p-STAT3 in RN5 cell growth, RNA-seq analysis was performed after treatment with JSI-124.RN5 cells were treated by JSI-124 with 500 or 750 nM for 24 h.Our results showed that both 500 and 750 nM JSI-124 significantly altered the gene expression profile at 24 h compared with that in the DMSO group (Padj <0.05, >twofold change in expression).The RNA-seq data showed that 973 genes were upregulated and 658 genes were downregulated following treatment with 750 nM JSI-124 for 24 h, while 685 genes and 304 genes were upregulated and downregulated, respectively, in the 500 nM group.All altered genes (Padj <0.05, >twofold change in expression) are presented in the volcano plot after RN5 cells were treated with 750 nM for 24 h (Figure 2A).A total of 11,358 genes expressed in the DMSO, 500 nM, and 750 nM JSI-124 treatment groups were grouped into 12 clusters using Mfuzz clustering (Supporting Information: Table S2).The genes in clusters 11 and 12 showed an increasing trend after treatment with 500 and 750 nM JSI-124, respectively.
The genes included in clusters 11 and 12 were combined to perform KEGG analysis.The KEGG results showed that autophagy and mammalian target of rapamycin signaling pathways were enriched (Figure 2B).
GSEA was performed to investigate the pathways activated or suppressed by JSI-124 treatment.GSEA of all genes expressed in 750 nM of JSI-124 using the Hallmark database showed that the pathways, such as E2F targets, MYC targets V1/V2, and G2M checkpoint were suppressed compared with RN5-DMSO-treated cells (Figure 2C).Whereas, pathways related to hypoxia, tumor necrosis factor signaling via NFκB, p53, and apoptosis were activated (Figure 2D).These pathways were also suppressed and activated in 750 nM JSI-124 treatment group compared with 500 nM treatment group (Supporting Information: Figure S3).

| JSI-124 induces caspase-dependent apoptosis
Previous studies have shown that JSI-124 induces G2/M cell-cycle arrest in various cancer cells. 12,13Similarly, our results show that JSI-124 induced G2/M cell-cycle arrest in RN5 cells (Supporting Information: Figure S4).Our RNA-seq analysis demonstrated the activation of apoptotic pathways caused by JSI-124 treatment at both 500 and 750 nM.We elucidated further whether apoptosis occurred in murine RN5 and AB12 cells.Flow cytometry was used to assess the JSI-124-induced apoptosis in AV-PI cells (Figure 3A).AV-PI staining indicated that treatment with 250, 500, and 750 nM JSI-124 increased the percentage of AV-and PI-positive cells.A total of 750 nM JSI-124 induced the highest levels of apoptosis in early and late stages (Figure 3B).To test whether JSI-124-related apoptosis was caspase-dependent, the expression of caspase 3 was examined.
The expression levels of full-length and cleaved caspase 3 were detected by western blot analysis.The expression level of cleaved caspase 3 increased with the dose after JSI-124 in 48 h.Additionally, the expression of cleaved PARP and Bax increased.Bcl-xL expression decreased in a dose-dependent manner in RN5 and AB12 cells (Figure 3C).

| JSI-124 triggers autophagy
Mfuzz clustering and KEGG enrichment analysis showed that JSI-124 treatment may induce the activation of the autophagy pathway.To observe autophagy induced by JSI-124 in murine RN5 and AB12 cells, TEM was used to examine the accumulation of autophagosomes in the cells after JSI-124 treatment.Autophagosomes accumulated with 750 nM JSI-124 treatment for 48 h (Figure 4A).Western blot analysis also showed that LC3b-II, a marker of autophagy, increased in a dose-and time-dependent manner, indicating autophagosome formation (Figure 4B).Additionally, RN5 and AB12 cells stably expressing RFP-GFP-tagged LC3b were used to monitor autophagosome formation.8][29] Activated STAT3 is also involved in MPM.However, the exact mechanism of STAT3 activation in MPM has not yet been elucidated.The active form of STAT3 (p-Tyr705 STAT3), located in the nucleus, has been observed in more than 50% of the archived MPM cases. 30Furthermore, low levels of PIAS3, a negative regulator of active STAT3, are associated with increased STAT3 activation and poor patient survival. 31I-124 is a dual inhibitor of JAK2 and STAT3. 9Although almost all studies have reported that JSI-124 inhibits the phosphorylation of STAT3 (Tyr), 10,11 one exception is that a low concentration of JSI-124 resulted in anti-tumorigenic activity, independent of the phosphorylation of STAT3 or Janus kinase (JAK2). 12JSI-124 inhibited both p-STAT3 and p-JAK2. 11Actin filaments are another target of JSI-124. 32However, whether JSI-124 can serve as a potential therapeutic agent for MPM remains unknown.In this study, we demonstrate the potential of JSI-124 to induce cytotoxicity in murine MPM by inhibiting the activation of STAT3.
Autophagy relies on the lysosomal pathways to degrade cytoplasmic proteins and organelles.It has a dual nature that can lead to the death of tumor cells, and also promote tumor development. 33,34Excessive activation of autophagy induces the occurrence and development of MPM. 35,36In addition, autophagy activation may lead to resistance to MPM chemotherapy. 37,38In the present study, we found that JSI-124 induced apoptosis and activated autophagy in MPM in a dose-dependent manner.Autophagy induced by JSI-124 in MPM may impair its apoptotic effect of JSI-124 on tumor cells.The phenomenon of JSI-124 promoting MPM cell apoptosis was amplified after cotreatment with an autophagy inhibitor.
Our RNA-seq data analysis revealed that 500 and 750 nM JSI-124 treatment induced E2F target genes, G2/M checkpoint, and MYC target V1 and V2 pathway suppression.Other STAT3 inhibitors, such as atovaquone and pyrimethamine, also induce the E2F target and the G2/M pathway in MPM. 39We also found that MYC targeted the V1 and V2 pathways and could be effectively suppressed by JSI-124, which has not been previously reported.Suppression of the pathways of E2F target genes, G2/M checkpoint, and MYC targets the V1 and V2 pathways may explain cell growth.
Although nivolumab plus ipilimumab has been proven beneficial in MPM treatment, MPM is still considered an immune-cold tumor, and only a small proportion of people benefit from it. 8Our research, along with other studies, has revealed that the activation of inflammatory response genes and pathways, including the nuclear factor kappa B (NF-kB) signaling pathway, occurs following the treatment with STAT3 inhibitor. 39,40Activated STAT3 plays an important role in converting old tumors into active tumors and in immune evasion. 41,42Therefore, JSI-124 shows potential in inhibiting immune evasion and reducing the immunosuppressive microenvironment.

2. 5 |
RNA extraction, complementary DNA (cDNA) library construction, and transcriptome sequencing RN5 cells were seeded in six-well plates at a density of 2 × 10 5 cells/ well and incubated at 37°C for 24 h.Cells were treated with vehicle control (dimethyl sulfoxide [DMSO] 0.1%) and various doses of JSI-124 (500 and 750 nM) for 24 h.Each group contained three biological replicates in which cells from three individual wells were pooled together at one time.

C57BL/ 6
J mice (weight, 20-25 g; age: 6 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.RN5 cells (5 × 10 6 cells in 100 μL PBS) were injected subcutaneously into the dorsal flank of each mouse.Body weight and tumor growth were measured daily.Tumor growth was calculated using the following formula: (L×W 2 )/2, where L is the length and W is the width.When the tumor volume reached 60 mm 3 , the mice were divided into four groups.Control animals were injected with solvent (10% DMSO diluted in PBS), whereas treated animals were injected with JSI-124 (1 mg/kg/day) in 10% DMSO in PBS, chloroquine (CQ) (25 mg/kg/ day) in 10% DMSO in PBS, or JSI-124 (1 mg/kg/day) combined with CQ (25 mg/kg/day) in 10% DMSO in PBS and administered intraperitoneally with 100 μL of vehicle or drug once daily for 21 consecutive days.Mice were euthanized when the tumors in the control group grew to 15 mm in diameter.Tumor samples were collected and fixed in a 10% formalin-neutral buffer solution.
Autophagic flux was used to further examine the autophagy induced by JSI-124 using Bafilomycin A1(Baf-A1) or CQ, which block the downstream steps of autophagy.Cotreatment of RN5 and AB12 cells with CQ or Baf-A1 and JSI-124 increased the conversion LC3b-II and accumulation (Figure 5A,B).RFP is more stable than GFP in the acidic pH of the lysosomal compartment, thereby RFP-LC3 can be used to identify both autophagosomes and autolysosomes.Red F I G U R E 1 JSI-124 suppressed cell proliferation of murine RN5 and AB12 cells in vitro by specifically targeting p-signal transducer and activator of transcription 3 (p-STAT3).(A) Cell viability of RN5 and AB12 cells treated with JSI-124 for 48 h.(B) IC50 of JSI-124 in RN5 and AB12 cells for 72 h.Data are shown as mean ± standard deviation from three independent experiments.(C) Expression levels of p-STAT3/STAT3 and p-Janus kinase 2 (p-JAK2)/JAK2 were detected in RN5 and AB12 cells treated with various concentrations of JSI-124 for 48 h by western blot.Data are presented as the means ± standard deviation from three independent experiments.*p < 0.05 and **p < 0.01 versus the DMSO group.dots in the merged images that do not overlay the green dots indicate autolysosome formation. 22Many red dots and yellow signals were observed in the merged images after JSI-124 treatment for 48 h, demonstrating that JSI-124 increased autophagic flux (Figure 5C).

3. 5 |
CQ enhanced the antitumor activity induced by JSI-124 in vitro and in vivoSubsequently, we determined whether the inhibition of autophagy enhanced apoptosis in JSI-124-treated RN5 and AB12 cells.

F I G U R E 2
Analysis of the transcriptome differentially expressed genes and signaling pathway enrichment.(A) Volcano plot of differentially expressed genes in RN5 cells with 750 nM JSI-124 treatment versus DMSO group.Each dot represents a single gene.Black dots mean no significant differentially expressed genes (DEGs) between 750 nM JSI-124 treatment and the DMSO group, blue dots mean downregulated genes, and red dots mean upregulated genes.(B) Bar plot of biological processes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of different categories of clusters 11 and 12. (C) Enrichment plot of suppressed signaling pathways of hallmark E2F target, G2M checkpoint, and MYC targets V1 and V2.(D) Enrichment plot of activated signaling pathways of hallmark hypoxia, p53, apoptosis, and TNFA signaling via NFκB.F I G U R E 3 Apoptosis induced by JSI-124 treatment for 48 h.(A) Apoptosis was detected using flow cytometry after staining RN5 and AB12 cells with fluorescein isothiocyanate-labeled annexin V and PI.(B) The percentage of RN5 and AB12 cells undergoing apoptosis induced by JSI-124 treatment for 48 h in three separate experiments.(C) Western blot analysis of PARP, Bcl-xL, Bax, and Caspase 3. *p < 0.05 and **p < 0.01 versus the DMSO group.As shown in Figures 6A,B, JSI-124 significantly inhibited the proliferation of RN5 and AB12 cells pretreated with autophagy inhibitors (CQ or Baf-A1).Additionally, PI-AV staining indicated that cotreatment of JSI-124 with autophagy inhibitors (CQ or Baf-A1) markedly increased apoptosis (Figure 6C,D).These results indicate that autophagy induced by JSI-124 treatment plays a protective role.To determine the potential therapeutic effects of JSI-124, tumor growth in mice was investigated in vivo.The tumor volume was markedly reduced after four injections of JSI-124.To confirm that F I G U R E 4 JSI-124 induces autophagy of RN5 and AB12 cells.(A) Transmission electron microscopy (TEM) images of RN5 and AB12 cells treated with DMSO (0.1%) or JSI-124 (750 nM) for 48 h.Arrows indicate autophagosomes.The quantification of autophagic vacuoles is presented by counting their numbers in at least 10 randomly chosen areas (***, p < 0.01) (Scale bar: 2.5 μm) (B) Western blot analysis to detect protein levels of LC3b and GAPDH for time and dose dependency.(C) Fluorescence images of GFP-LC3b puncta in RN5 and AB12 cells treated with JSI-124 for 48 h.The number of GFP-LC3b dots in each cell was quantified.Data are presented as mean ± SD from three independent experiments.**p < 0.05 and **p < 0.01 versus the DMSO group (Scale bar: 10 μm).

F
I G U R E 5 JSI-124 induces autophagy flux in RN5 and AB12 cells.(A) A total of 20 μg protein was loaded to detect LC3b in RN5 cells and AB12 cells pretreated with CQ (30 μM), followed by exposure to JSI-124 (500 nM) or DMSO for another 48 h.(B) A total of 20 μg protein was loaded to detect LC3b in RN5 cells and AB12 cells pretreated with Baf-A1 (4 nM), followed by exposure to JSI-124 (500 nM) or DMSO for another 48 h.(C) Fluorescence images of RFP-GFP-LC3b puncta in RN5 and AB12 cells treated with JSI-124 (500 and 750 nM) for 48 h and untreated RN5 and AB12 cells.The quantification of acidified autophagosomes (GFP -RFP + ) versus neutral autophagosomes (GFP + RFP + ) per cell was performed for each experimental condition.The data are represented as the mean ± standard deviation (SD) from three independent experiments (N.S, not significant;*, p < 0.05).Scale bar: 10 μm.F I G U R E 6 (See caption on next page).autophagy was induced by JSI-124 during apoptotic cell death, CQ was used to prevent autophagy in vivo.As shown in Figure 6E,F, CQ significantly enhanced JSI-124-induced tumor growth.To evaluate the side effects of JSI-124, the body weights of the mice were monitored throughout the study.As shown in Figure 6G, no obvious body weight loss was observed in mice treated with JSI-124, CQ, or JSI-124 combined with CQ.This result demonstrates that doses of JSI-124 and JSI-124 combined with CQ used in this study did not show significant systemic toxicity in vivo.Previous in vitro studies have shown that JSI-124 enhances apoptosis after the inhibition of autophagy.To confirm this result in vivo, TUNEL analysis was performed on tumor sections.The tumors treated with JSI-124 and CQ showed an increased number of TUNEL-positive apoptotic cells (Figure 6H).

4 |
DISCUSSION This study demonstrated that JSI-124 induces autophagic and apoptotic cell death in murine RN5 and AB12 murine MPM cells via a STAT3-dependent pathway.Further analysis indicated that the suppression of autophagy induced by JSI-124 enhanced apoptotic cell death both in vitro and in vivo.These results indicate that JSI-124 is a potential therapeutic agent for the treatment of mesothelioma.STAT proteins are a group of cytoplasmic transcription factors and can be activated by various extracellular signaling proteins, such as cytokines, growth factors, and hormones.STAT proteins perform signal transduction in the cytoplasm and function as transcription factors in the nucleus.Aberrant activation of STAT3, resulting from the phosphorylation of tyrosine 705 within the transcription activation domain, is involved in tumor cell development, progression, drug or radiation resistance, and poor clinical prognosis.

F I G U R E 6
Autophagy induced by JSI-124 plays a protective role in RN5 cells and the antitumor effects of JSI-124 in mice.(A-B) Inhibition of autophagy enhances JSI-124-induced suppression of cell proliferation in RN5 (A) and AB12 cells (B).(C-D) Inhibition of autophagy enhances JSI-124-induced apoptosis in RN5 (C) and AB12 cells (D).The antitumor effects of JSI-124 injected with RN5 cells in vivo at a dose of 1 mg/kg/ day, CQ 25 mg/kg/day, and JSI-124 1 mg/kg/day in combination with CQ 25 mg/kg/day.The mice were dosed intraperitoneally with a vehicle, CQ (25 mg/kg/day), JSI-124 (1 mg/kg/day), and JSI-124 (1 mg/kg/day) combined with CQ (25 mg/kg/day) once daily for 22 consecutive days.The mice were euthanized 21 days after the last dose of treatment.Tumor volume (E), tumor weight (F) body weight (G) and were measured.Each group has five to six mice.(H) Representative histological micrographs of TUNEL staining.TUNEL staining was semiquantitatively evaluated by positive cells.All data are presented in mean ± SD. *p < 0.05 and **p < 0.01 versus the solvent group.#p < 0.05 and ##p < 0.01 versus the JSI-124 group.Scale bar: 100 μm.In conclusion, JSI-124 induced apoptotic and autophagic cell death and inhibited cell proliferation and cell-cycle arrest in a STAT3dependent manner in murine MPM.The combination treatment of CQ and JSI-124 inhibited cell proliferation with high efficacy and low toxicity.Suppression of E2F target genes, G2/M checkpoint, and MYC target V1 and V2 pathways can be used to explain cell-cycle arrest.Our results support the notion that JSI-124 inhibits cell proliferation and tumor growth by inducing autophagy and apoptosis in murine malignant mesothelioma.