Anti-tumor effects and mechanism of the histone demethylase inhibitor GSK-J4 in non-small cell lung cancer cells

Abstract

Lung cancer is one of the leading causes of death worldwide. Cancer metastases are responsible for 90% of cancer-related deaths. In view of the high incidence and mortality of lung cancer, there is still an urgent clinical need to improve the early diagnosis of lung cancer and explore new therapeutic methods and targets to improve the prognosis of patients. By targeting promoter or enhancer regions of related genes, histone methylation modification dynamically regulates gene expression and activation of signaling pathways, and is involved in mediating malignant biological processes such as proliferation, invasion and metastasis of tumor cells. GSK-J4 is a novel small molecule inhibitor of the JMJD3 and UTX family of selective histone demethylases, which shows good anticancer activity against various types of tumors by inhibiting H3K27me3 demethylation. The aim of this study was to investigate the effects of GSK-J4 on proliferation, migration, invasion and epithelial-mesenchymal transformation (EMT) of TGF-β1-induced non-small cell lung cancer (NSCLC) cell lines A549 and H1299. The effect of GSK-J4 on cell proliferation was detected by CCK8, clonal formation assay, immunofluorescence and flow cytometry. The effects of GSK-J4 on the migration and invasion of A549 and H1299 cells induced by TGFβ1 were examined by wound healing assay, Transwell migration assay, and then, the expression changes of related markers were detected by RT-qPCR and western blot. Finally, GSK-J4 was verified to inhibit tumor growth in vivo by constructing a mouse model of tumor implantation in situ, and observed its effectiveness and safety. GSK-J4 inhibited proliferation and promoted apoptosis of A549 and H1299. GSK-J4 inhibited EMT, invasion and migration of TGFβ1-induced NSCLC. GSK-J4 inhibits EMT, invasion and migration of TGFβ1-induced NSCLC through the classical Wnt/β-catenin signaling pathway. In situ tumor model, GSK-J4 administration alone effectively reduced tumor growth in nude mice, with the tumor size being notably less than in the control group. IHC analysis showed that the expression of Ki-67 in GSK-J4 administration group was lower than that in control group. HE staining showed that GSK-J4 had no significant effect on the histopathology of heart, liver, lung, kidney and other major organs of mice. GSK-J4, an inhibitor of histone demethylase, elevated the level of H3K27me3 by suppressing JMJD3/UTX activity, thus curbing NSCLC cell growth and encouraging cell death. Concurrently, GSK-J4 played a role in preventing TGFβ1-triggered EMT, invasion, and migration. Consequently, targeting histone methylation modification and the small molecule inhibitor GSK-J4 is anticipated to be an effective treatment strategy and a novel method for NSCLC.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12032-025-03185-3.

Introduction

Lung cancer is a malignant tumor originating from epithelial cells in the bronchi, and is one of the leading causes of death from diseases worldwide. According to the Global Cancer Statistics 2022, there were nearly 2.5 million new lung cancer cases globally, making it the most commonly diagnosed cancer and the main cause of cancer-related deaths, with over 1.8 million new deaths [1, 2]. In China, the incidence and mortality rates of lung cancer are still increasing year by year [3]. Among them, non-small cell lung cancer (NSCLC) is the main pathological type, accounting for about 85% [4]. Due to the lack of specific clinical manifestations in early lung cancer, most lung cancers are diagnosed at an advanced stage, with a 5-year survival rate for advanced NSCLC less than 15% [5, 6], and a median overall survival time for metastatic NSCLC less than 3 years [7, 8]. Existing research shows that targeted therapy based on the epidermal growth factor receptor (EGFR) gene mutations and their variants has made significant progress in NSCLC [9–11]. The use of tyrosine kinase inhibitor (TKI) drugs in those patients with EGFR mutations has contributed to a better response to therapy, but the side effects gradually exhibited by these drugs limit their application [12]. Other treatments (immunotherapy, etc.) applied alone or in combination, there are still issues such as low response rates, acquired or intrinsic resistance, and immune-related adverse events. Therefore, given the high incidence and mortality rates of lung cancer, improving early diagnosis and exploring new treatment modalities and targets, and improving patient outcomes, still has urgent clinical needs.

Epithelial-mesenchymal transition (EMT) is the process by which epithelial cells transform into a mesenchymal cell phenotype, losing their original cell-cell adhesion and polarity while acquiring the invasive, migratory, and ECM-producing characteristics of mesenchymal cells. EMT plays a critical role in tumor development and progression, including enhancing tumor invasion and infiltration capabilities, promoting tumor cell stemness, increasing drug resistance in tumor cells, and accelerating tumor metastasis [13–15]. The metastasis or spread of cancer from the primary site to form secondary tumors in distant locations accounts for 90% of cancer-related deaths [16]. Therefore, inhibiting EMT may improve the disease progression of NSCLC and enhance patient prognosis.

Epigenetic modification is the process of regulating gene expression without changing the DNA sequence, which has become one of the hallmarks of cancer [17]. There is increasing evidence that dysregulation of epigenetic modification enzymes is associated with the occurrence and development of cancer, and that epigenome-related molecular therapies can improve cancer symptoms. Histone methylation is an important process in epigenetic regulation. The four lysine residues in the N-terminal tail of the conserved histone (K4, K9, K27, K36) is the main target of specific histone methyltransferase and demethylase. Changes in methylation can dynamically regulate gene expression and activation of signaling pathways through targeted binding of related gene promoters or enhancers, and participate in mediating malignant biological processes such as tumor cell proliferation, invasion and metastasis [18–22], especially in breast cancer [23, 24], NSCLC [25], ovarian cancer [26] and prostate cancer [27]. Epigenetic proteins EZH2 and JMJD3/UTX are two enzymes that control the methylation level of histone H3 at the K27 site (H3K27) and are major components of cancer aggressivity and EMT [28]. Studies have shown that lower levels of H3K27 trimethylation are associated with higher tumor aggressiveness and/or poor disease-free survival (DFS), that low expression of H3K27me3 is a strong and independent predictor of poor differentiation of NSCLC cells and shortened cancer-specific survival, and that high expression of H3K27me3 is associated with prolonged overall survival of NSCLC [29].

GSK-J4 is a novel selective histone demethylase JMJD3 and UTX family of small molecule inhibitors, by inhibiting H3K27me3 demethylation has shown good anticancer activity against many types of tumors, such as breast cancer, lung cancer, liver cancer, prostate cancer, colorectal cancer, thyroid cancer and leukemia [30–39]. The study also pointed out that the anti-tumor effect of GSK-J4 is reflected in many aspects, including inhibiting the heterogeneous immune response of proliferation, migration and invasion of tumor stem cells and sensitivity with radiotherapy and chemotherapy, etc. However, there are few studies on how GSK-J4 plays a role in NSCLC, and more in-depth exploration of the role of GSK-J4 is needed.

The aim of this study was to investigate the effects of GSK-J4 on the proliferation, migration, invasion and EMT of TGF-β1-induced NSCLC (A549 and H1299) cells, and explore its potential mechanism, providing evidence that GSK-J4 may be considered as a promising drug for the treatment of NSCLC.

Materials and methods

The main reagent

GSK-J4 (T3100) and TGFβ1 (TMPY-02638) was purchased from Targetmol, Shanghai, China. GSK-J4 was dissolved with DMSO, with an initial concentration of 10mmol/l; TGFβ1 was dissolved in sterile deionized water at an initial concentration of 0.1 mg/ml. Dilute with medium to desired concentration when using.

Cell culture

The A549(RRID: CVCL_0023) and H1299(RRID: CVCL_0060) cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and stored in the Second Affiliated Hospital of Harbin Medical University. We confirmed that mycoplasma testing has been done for the cell lines used and strains were identified (DNA was extracted with Axygen’s genome extraction kit, amplified by 21-STR amplification protocol, and the STR locus and sex gene Amelogenin were detected on ABI 3730XL genetic analyzer, Matching degree 100%)(Supplementary Figure). The cells were removed from the − 80℃ refrigerator, quickly thawed in 37℃ water bath, neutralized with complete medium, the A549 cells were cultured in DMEM/F/12 medium (Gibco, USA), H1299 cells were maintained in RPMI 1640 medium (Gibco, USA), supplemented with 10% fetal bovine serum (Sigma, Merck KGaA, Germany), 100U/mL penicillin and 100 µg/mL streptomycin (Kaiji Biotech Company, Jiangsu, China), centrifuged at 1000 rpm for 3 min, discarded supernatant, and then re-suspended in a 6 cm dish with complete medium and cultured in a 37℃ 5% CO2 cell incubator. It is passed down every 2–3 days.

Cell viability assay

The cell viability was measured by CCK8 (GLPBIO, USA) method. The density of 5000cells per well was evenly spread in 96-well plates overnight, and the cells were attached to the wall the next day and replaced with the medium with medicine. Blank control group/control group/DMSO control group and GSK-J4 group with different concentrations (1, 2, 4, 8, 16, 32µmol/l) were administered for 24 h, 48 h and 72 h, respectively [40]. Then culture medium: CCK8 = 10:1 culture medium was changed, and the absorbance value at 450 nm wavelength was measured by enzymoleter after incubation for 2 h in the dark. Cell viability (%) was calculated according to the formula: = (experimental group - control group)/(control group - blank group) *100%.

Immunofluorescence co-staining

The 4*10⁴cells per well were evenly distributed in confocal small dishes, treated for 72 h after adhesion, fixed with 4% paraformaldehyde, closed with 5% goat serum for 1 h, added with primary antibody, incubated with 4 overnight, washed with TBST, added with secondary antibody, incubated for 1–1.5 h, sealed with DAPI tablets after TBST washing. Fluorescence staining was observed and photographed under laser confocal microscopy (ZEISS LSM710).

Cell apoptosis detection (flow cytometry)

According to the operation steps of the Annexin-V/PI Double Staining Apoptosis Detection Kit (Beyotime, Shanghai, China). Collect the processed A549 and H1299 cells, resuspend them in PBS, and take 5–10*10⁴ resuspended cells. Centrifuge the cells and discard the supernatant. Add 195 µl of Annexin V-FITC binding buffer, followed by 5 µl of Annexin V-FITC and 10 µl of PI staining solution. Incubate the mixture at room temperature in the dark for 10–20 min. After filtering the cells through a cell strainer, analyze them using a flow cytometer (BD Accuri C6, USA) within 1 h. Perform data analysis using FlowJo software (version: 10.8.1) [41].

Real-time fluorescence quantification PCR(RT-qPCR)

Total RNA was extracted from the cells using RNA extraction kit, and was reverse-transcribed into cDNA. Primers were designed according to the Prime-blast website, and fluorescence quantitative PCR was performed (Real-time fluorescence quantitative PCR instrument, BIO-RAD Company, USA). GAPDH was used as the internal reference 2^-ΔΔCt method for statistical analysis [42]. All parameters repeated at least three times. For detailed primer sequences, refer to Supplementary Table 1.

Wound healing test

At the back of the 6-well plate, each hole was marked with two evenly spaced horizontal lines, and then inoculated on the identical plate at a rate of 1*10⁶ cells per hole. After the cells were attached to the wall for about 24 h, the cell layer was scraped from one end of each hole to the other end with a 200ul sterile pipette gun, making it perpendicular to the horizontal line, and the cell 3 was washed with PBS.The image of scratch width was captured under an inverted microscope (Olympus IX51) (0/24/48 hours). The same part was photographed each time. The experiment was repeated three times and calculated according to the following formula: cell mobility% = (0 h scratch width − 24/48 hours scratch width)/0 h scratch width*100%.

Transwell cell migration assay

The migration ability of A549 and H1299 cells in good condition were detected by transwell assay and diluted with serum-free medium. The density of 2*10⁴cells per pores was inoculated into the upper chamber of transwell chamber, and the lower chamber was added with complete medium containing 10% FBS and containing TGFβ1 and TGFβ1 + GSK-J4, respectively. After 48 h, the liquid in the upper chamber was discarded, and the surface of the upper chamber was gently wiped with a wet cotton swab, and fixed in 4% paraformaldehyde for 30 min, PBS moisten and wash twice, then place in crystal violet dye for 30 min, rinse, dry naturally, and take photos under the microscope.

Angiogenesis test

HUVEC cells in the logarithmic growth phase and in good growth condition were taken for the angiogenesis experiment. On the day before the angiogenesis experiment, HUVEC cells with good growth state in logarithmic growth stage. Collect the culture medium of A549 cells in advance as the conditioned medium and the conditioned medium treated with GSK-J4. Pre-cool the 24-well plate and 200 µl gun head, and melt the matrix glue slowly at 4℃ and 60 µl matrix glue was evenly spread on 24-well plate for 4℃ overnight. On the second day, the 24-well plates were first placed in 37℃ incubators for matrix gel solidification, HUVEC cells were replaced with serum-free medium and starved for 6 h, then the medium was abandoned, digested by pancreatic enzymes, centrifuged at 1000 rpm for 3 min, and the cells were suspended again, pressing 1*105cells/well count, 500ul/well, were divided into A549 + HUVEC and A549 + GSK-J4 + HUVEC administration group, which were placed back in 37℃ incubator for further culture. 4 h later, the experiment was repeated three times under the microscope for statistical analysis.

Western blot anglysis

A549 and H1299 in good condition were passed into 6 cm dishes, and after 24 h, they were replaced with conditioned medium containing 10ng/ml TGFβ1 and TGFβ1 + GSK-J4, and the cells were treated for 72 h. Cells were lysed using a RIPA lysis buffer containing 1% PMSF. The protein was quantified by BCA protein kit. The proteins were isolated by SDS-PAGE electrophoresis and transferred to PVDF membranes (transfer mode 1–2 h). Closed with 5% skim milk for 1 h, incubated with primary antibody and incubated at 4℃ overnight. On the second day, the film was washed with TBST and incubated at room temperature for 1 h with secondary antibody. The film was washed with TBST for 3 times, 10 min each time, and the film was developed. ImageJ performs gray value analysis of the strip [43]. All parameters repeated at least three times. The antibody information used in the research is as follows: Anti-Ki67(Abcam, RRID: AB_302459), Anti-Bax(MCE, RRID: AB_3102269), Anti-Bcl-2(MCE, RRID: AB_3102133), Anti-Wnt3a(MCE, RRID: AB_3676593), Anti-MMP2(Abways, RRID: AB_3696820), Anti-MMP9(Selleck, RRID: AB_3696822), Anti-β-catenin(Sellcek, RRID: AB_3696821), Anti-E-cadherin(CST, RRID: AB_3696827), Anti-N-cadherin(CST, RRID: AB_3696828), Anti-Fibronectin(CST, RRID: AB_3696826), Anti-CyclinD1(Selleck, RRID: AB_3696823), Anti-Birc5(Selleck, RRID: AB_3696825), Anti-β-Actin(Huaan Biotechnology Co.,Ltd., RRID: AB_3073045).

In vivo experiment

The tumor-bearing mouse model was constructed. BALB/c nude mice, purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., LTD, 4–6 weeks old, 16–18 g, acclimated for one week. A549 cells (about 5*10⁶cells/ml cells) were inoculated subcutaneously with 200 µl PBS in the anterior axilla. After the cells were inoculated, the mental state, activity status and response to external stimuli of each group of nude mice were observed daily. When the subcutaneous nodules grew to 60-100mm³, they were randomly divided into control group and GSK-J4 administration group, with 4 mice in each group. The control group was given equal concentration of DMSO, and the experimental group was given 100 mg/kg GSK-J4 solution by intrabitoneal injection, once every other day, a total of 5 times [31]. The changes of body weight and tumor volume in nude mice were recorded in detail. The long diameter (a) and short diameter (b) of tumor nodules were measured with vernier calipers. The tumor volume was calculated according to the formula : Tumor volume (v) = 0.5×a×b², and the tumor growth curve was drawn.

The experiment was terminated on the 3rd day after the last administration, and mice were euthanized, tumor body dissection was performed immediately, tumor weight was weighed and recorded, tumor tissue was cut into several small pieces, part of the paraformaldehyde was fixed for HE staining and immunohistochemical analysis, and part of the obtained internal organs were preserved at −80℃. The obtained internal organs (lung, heart, liver and kidney) were fixed in paraformaldehyde, dehydrated and embedded in paraffin, and pathological sections were made, and routine HE staining was performed to observe the presence of lesions and immunohistochemical analysis. This animal study was reviewed and approved by the Medical Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (YJSDW2023-023). All methods were performed in accordance with the relevant guidelines and regulations. All experiments were conducted in accordance with the ARRIVE guidelines.

Statistic analysis

GraphPad Prism 10.0 software was used for statistical analysis. All experiments were repeated at least three times. The two-tailed T-test was used to compare the two groups. Comparisons between groups were performed using one-way analysis of variance with Bonferroni’s post hoc test or unpaired two-tailed Student’s t-test. P<0.05 was defined as having a statistically significant difference (*P < 0.05, **P < 0.01, ***P < 0.001).

Results

GSK-J4 inhibited the proliferation of A549 and H1299 cells

NSCLC cells A549 and H1299 were treated with different concentrations of GSK-J4 (0, 1, 2, 4, 8, 16, 32µmol/l), respectively, and the cell viability was observed for 24 h, 48 h and 72 h after drug treatment. The absorbance value at 450 nm was detected by enzyme labeling Analysis results show that is shown in Fig. 1A: GSK-J4 reduced the proliferation of A549 and H1299 cells in a time - and dose-dependent manner. The IC50 values of 24 h, 48 h and 72 h in A549 cell line were 18.98µmol/l, 2.654µmol/l and 1.370µmol/l, respectively. The IC50 values of 24 h, 48 h and 72 h in H1299 cell line were 11.60µmol/l, 4.559µmol/l and 3.134µmol/l, respectively. Therefore, we chose 4µmol/l (A549) and 8µmol/l (H1299) for subsequent experiments. The results of the colony formation experiment showed that GSK-J4 reduced the colony-forming ability of A549 and H1299 cells (Fig. 1B), which further confirmed the ability of GSK-J4 to inhibit the proliferation of NSCLC cells.

Fig. 1.

Effect of GSK-J4, a small molecule inhibitor of histone demethylase, on proliferation and apoptosis of NSCLC cell line. (A) Cell proliferation experiments. A549 and H1299 were treated with control group, DMSO and GSK-J4 at different concentrations, respectively, and the absorbance values were determined at 24 h, 48 h and 72 h. (B) Colony formation experiments. DMSO was compared with GSK-J4 to observe the colony formation ability. (C) Immunofluorescence co-staining DSMO and GSK-J4 were applied to A549 and H1299, respectively, and the expression and localization of Ki-67 were observed 48 h later. (D, E) RT-qPCR and western blot were used to detect the expression of apoptosis-related markers, respectively. (F) Flow cytometry analysis after DMSO and GSK-J4 were treated for 72 h, apoptosis was detected by flow cytometry analyzer. (G) Angiogenesis test. Effect of GSK-J4 on angiogenesis of vascular endothelial cells HUVEC. Scale: 200 μm. FlowJo, ImageJ, and GraphPad Prism were used to analyze the data.(Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001)

Then, we performed immunofluorescence co-staining to detect the localization and expression of Ki-67 after GSK-J4 acted on A549 and H1299 cells, and the results showed that GSK-J4 reduced the expression of Ki-67, a cell proliferation marker (Fig. 1C).

In addition, we detected the changes of mRNA and protein levels of proliferation and apoptosis-related indicators by RT-qPCR and Western blot, respectively, and the results confirmed that after GSK-J4 administration, the expression of anti-apoptotic genes was inhibited, while the expression of pro-apoptotic genes was up-regulated (Fig. 1D, E). Finally, we tested the effect of GSK-J4 on apoptosis by flow cytometry (Fig. 1F), which also confirmed the above results.

Tumor angiogenesis is one of the signature characteristics of tumors, which provides energy for the continuous division and proliferation of tumor cells, and is an important condition for tumor development, invasion and metastasis. Several histone demethylases, including KDM2A, KDM2B, KDM4D, KDM5B, and KDM7A, have been shown to play a role in hypoxia-mediated tumor angiogenesis. Liu et al. showed that KDM6B mediates H3K27me3 demethylation, and is involved in regulating VEGFA expression and tumor angiogenesis [44]. Therefore, we treated vascular endothelial cells HUVEC with A549 + GSK-J4 observe its regulation of angiogenesis. The results showed that after the effect of GSK-J4, we observed the changes in cell morphology and structure at 2 h, 4 h, 6 h, and 12 h respectively, and GSK-J4 significantly inhibited the tubulogenesis ability of HUVEC cells (Fig. 1G).

Therefore, we speculate that the histone demethylase inhibitor GSK-J4 plays a role in promoting apoptosis and inhibiting proliferation of NSCLC, which is expected to be a new direction for the treatment of NSCLC.

GSK-J4 inhibits the migration of A549 and H1299 cells induced by TGFβ1

Transforming growth factor β1 (TGFβ1) is a recognized strong inducer of EMT, and EMT plays an important role in the invasion and metastasis of tumors, which can affect the disease process and drug resistance to radiotherapy and chemotherapy. Therefore, in order to explore the role of GSK-J4 in the invasion and metastasis of NSCLC, A549 and H1299 cells were treated with TGFβ1(10ng/ml) for follow-up studies.

First, in order to study the effect of GSK-J4 on the migration and invasion ability of A549 and H1299 induced by TGFβ1, Fig. 2A observed the effect of GSK-J4 on migration in 2D structure of cells through wound healing experiment, and the results showed that GSK-J4 inhibited TGFβ1-induced cell migration. Subsequently, we performed transwell for migration. As shown in Fig. 2B, C, the number of A549 and H1299 cells migrated significantly increased after TGFβ1 treatment, while GSK-J4 significantly inhibited the migration of A549 and H1299 cells compared with the corresponding control group.

Fig. 2.

GSK-J4 inhibits the migration and invasion of A549 and H1299 cells induced by TGFβ1. (A) Wound healing experiments. Control group, TGFβ1 group, TGFβ1 + GSK-J4 group were set up respectively, and scratch widths at 0 h, 24 h and 48 h were observed to evaluate cell migration. (B, C) Transwell migration experiment. The ability of the cells to pass to the lower chamber was observed. (D-F) RT-qPCR and western blot. The mRNA and protein levels of mmp2/MMP2 and mmp9/MMP9, which are related to cell migration and invasion were detected. Scale: 200 μm.(Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. * control vs. TGFβ1; # TGFβ1 vs. TGFβ1 + GSK-J4)

Matrix metalloproteinases (MMPs) is a family of ECM-degrading peptidases, which is mainly related to the degradation and reconstruction of ECM, and has a positive contribution to the invasive and metastatic process of tumors [45, 46]. Therefore, we detected the changes of mRNA and protein levels of MMP2 and MMP9 by RT-qPCR and western blot. The results showed that TGFβ1 significantly increased the mRNA and protein levels of mmp2/MMP2 and mmp9/MMP9. This change was reversed after administration of GSK-J4, with reduced levels of mmp2/MMP2 and mmp9/MMP9 (Fig. 2D–F). MMP-2 and MMP-9 proteins are two key promotors that enhance the potential of tumor metastasis and invasion. Therefore, the histone demethylase inhibitor GSK-J4 is expected to be used in the treatment and intervention of clinical NSCLC by targeting two key targets.

GSK-J4 inhibits TGFβ1-induced EMT of A549 and H1299 cell lines

After treating A549 and H1299 cells with TGFβ1 (10ng/ml) for 72 h, RT-qPCR and western blot results showed that the expression of epithelial markers cdh1/E-cadherin decreased, while the expression of mesenchymal markers cdh2/N-cadherin and fn1/FN1 increased, indicating that TGFβ1 induced EMT in A549 and H1299 cells. Subsequently, we co-acted GSK-J4 with TGFβ1 on A549 and H1299, and GSK-J4 inhibited TGFβ1-induced EMT of A549 and H1299 cell lines (Fig. 3A–C).

Fig. 3.

GSK-J4 inhibits TGFβ1-induced EMT of A549 and H1299 cell lines. (A) RT-qPCR was used to detect EMT related markers, cdh1, cdh2, fn1. (B, C) Western blot analysis was performed to detect the expression changes of E-cadherin, N-cadherin and FN1 at the protein levels.(Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. * control vs. TGFβ1; # TGFβ1 vs. TGFβ1 + GSK-J4)

GSK-J4 inhibits TGFβ1-induced EMT of A549 and H1299 cell lines through the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway has been reported to have a significant impact on the maintenance of cancer metastasis and is one of the important pathways involved in the induction of EMT, which is associated with TGFβ1-induced cancer progression. Therefore, we investigated the effect of GSK-J4 on the Wnt/β-catenin pathway in TGFβ1-induced EMT in A549 and H1299 cells. We first detected the changes of mRNA levels of major markers in this pathway by RT-qPCR. The results showed that TGFβ1 significantly activated the classical Wnt/β-catenin signaling pathway, with upregulated levels of wnt3a and β-catenin, and downstream target genes birc5 and ccnd1. GSK-J4 reversed the effects of TGFβ1 (Fig. 4A). The same results were obtained when we used western blot to detect changes in the expression of markers related to protein levels (Fig. 4B, C).

Fig. 4.

GSK-J4 inhibits TGFβ1-induced EMT of A549 and H1299 cell lines via the Wnt/β-catenin signaling pathway. (A) Changes of markers associated with the Wnt/β-catenin signaling pathway at mRNA levels. (B, C) Western blot was used to detect the changes of markers related to Wnt/β-catenin signaling pathway at the protein level. (D)Expression and localization of β-catenin in A549 and H1299 cells induced by TGFβ1 by immunofluorescence staining. Scale bar : 100 μm. (Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. * control vs. TGFβ1; # TGFβ1 vs. TGFβ1 + GSK-J4)

To further verify that GSK-J4 exerts its effect by inhibiting the wnt/β-catenin signaling pathway, we also further observed the expression and localization of β-catenin in this process through immunofluorescence staining in A549 and H1299 cells, after the action of TGFβ1, the expression of β-catenin was promoted and the translocation of β-catenin from the cytoplasm to the nucleus was facilitated, while GSK-J4 inhibited its effect (Fig. 4D). Therefore, we hypothesized that GSK-J4 inhibits TGFβ1-induced EMT in A549 and H1299 cell lines via the wnt/β-catenin signaling pathway.

Subsequently, in order to further verify the function of the Wnt/β-catenin pathway in TGFβ1-induced A549 and H1299 progression, the Wnt pathway activator LiCl (20mmol/l) was used to activate the Wnt/β-catenin signaling pathway. As shown in Fig. 5A, B, GSK-J4 inhibited the migration of A549 and H1299 cells and the expression of MMP2 and MMP9 induced by TGFβ1, while LiCl reversed these effects (Fig. 5C–F). As shown in Fig. 5G–J, Licl also reversed the expression of β-Catenin, E-cadherin, FN1, CyclinD1, Birc5. Therefore, the above results support the anti-tumor effect of GSK-J4 on NSCLC cells by regulating Wnt/β-catenin signaling pathway to inhibit migration, invasion and EMT.

Fig. 5.

Restoration experiments were performed on Licl treated cells. (A, B) Transwell performed cell migration experiments. (C–F) Changes in the protein levels of MMP2 and MMP9, markers related to cell migration and invasion. (G–J) Western blot detection of changes in markers related to the Wnt/β-catenin signaling pathway at protein level. (Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. * control vs. TGFβ1;#TGFβ1 vs. TGFβ1 + GSK-J4; ^TGFβ1 + GSK-J4 vs. TGFβ1 + GSK-J4 + Licl)

GSK-J4 inhibits tumor growth in vivo

In order to verify the tumor inhibition effect of GSK-J4 in vivo, we subsequently constructed a xenograft tumor model with A549 cells. The mice were denecked and killed 3 days after the last dose. The tumor tissue, lung, heart, liver and kidney tissue of mice were extracted. Compared with the control group, intraperitoneal injection of GSK-J4 significantly inhibited tumor growth (Fig. 6A–D). There was no significant change in body weight between the two groups. Immunohistochemical analysis showed that the expression level of ki-67 in tumor tissues in the administration group was significantly decreased (Fig. 6E). Subsequently, we conducted a preliminary analysis on the safety of GSK-J4 after administration, and there was no significant change in HE staining of the heart, lung, liver and kidney tissues of the two groups of mice (Fig. 6F).

Fig. 6.

GSK-J4 inhibited tumor growth in nude mouse transplanted tumor model. (A) Construct a mouse transplanted tumor model. (B–D) Changes in tumor size, volume and body weight in two groups of mice. (E) Immunohistochemical analysis of tumor tissue. Scale: 100 μm. (F) HE staining results of heart, lung, liver and kidney of mice in two groups. Scale: 100 μm. (***P < 0.001)

The combination of GSK-J4 and cisplatin synergistically inhibited the proliferation of NSCLC cells

Platinum-based chemotherapy is the current standard treatment for lung cancer. Platinum-based compounds cause DNA damage by forming covalent adducts with cell DNA, leading to cell apoptosis and exert their anti-tumor activity. But platinum resistance remains a serious problem. Previous literature has reported that GSK-J4 can enhance the sensitivity of tumors to the chemotherapy drug cisplatin. Therefore, we investigated whether GSK-J4 could enhance the sensitivity of lung cancer cells to cisplatin. The results are shown in the figure below: When GSK-J4 and CDDP were used alone (Fig. 7A, B), the proliferation of both lung cancer cells (A549 and H1299) was inhibited with increasing drug concentration. When the two drugs were used in combination (Fig. 7C-D), cell proliferation was inhibited and the inhibitory effect was more pronounced. The combination index CI of the two drugs in combination was calculated using the Chow-Talalay method, and CI < 1 indicated that GSK-J4 and CDDP showed synergistic effect of the drugs in non-small cell lung cancer cells (Fig. 7E, F).

Fig. 7.

The combination of GSK-J4 and cisplatin synergistically inhibits the proliferation of NSCLC cells. (A, B, C, D) The effect of GSK-J4, CDDP and GSK-J4 + CDDP on A549 and H1299 cells for 48 h was detected by CCK8 method, and the OD value at 450 nm was measured by enzyme marker to draw the cell viability curve. (E, F) CompuSyn software was used to calculate the CI index of the combination of two drugs. CI < 1 indicated that the two drugs had combined effect; CI = 1 additive effect; CI > 1 antagonism

Discussion

NSCLC is one of the leading causes of cancer death worldwide. Rapid treatment continues to improve and advance, but the overall survival rate for advanced NSCLC remains low. Tumor cells engage in a complex ‘dialogue’ with surrounding tissues, cells, and the extracellular matrix environment, secreting various cytokines and activating signaling pathways that collectively drive tumor growth, immune evasion, and metastasis [47]. Cancer metastasis is a key marker of cancer deterioration, and EMT is considered to be the main mechanism of cancer metastasis and invasion [48]. EMT is also associated with the production of cancer stem cells within the primary tumor [49], and can also be involved in tumor chemotherapy resistance [50]. Therefore, inhibiting EMT during tumor progression is a very important strategy for the treatment and prevention of NSCLC, as well as for inducing cancer cell death [51].

In recent years, more and more evidence has shown that histone modification plays an important role in the occurrence and development of cancer. Histone methylation modification has become a key player in gene expression, cell cycle, aging, genomic stability, and nuclear structure regulation [52, 53]. For example, methylation of H3K4 and H3K79 is commonly associated with transcriptional activation, while methylation of H3K9 and H3K27 represses gene transcription [54]. Histone methylation modification is regulated by histone methyltransferase and histone demethylase. Dysregulation of histone methylation " writing proteins " and “erasing proteins” is closely associated with clinical outcomes in lung cancer patients through multiple cellular pathways related to proliferation, invasion, EMT, etc [55], the reversibility of these epigenetic aberrations makes them important targets for cancer treatment. The methylation status of histone H3K27 is regulated by methyltransferase EZH2, a core-catalytic subunit of the PRC2 complex that helps maintain cell identity cell cycle regulation and tumorigenesis, and demethylase JMJD3/UTX [56, 57]. EZH2 is highly expressed in a variety of solid human malignancies, including NSCLC, and overexpression of EZH2 is associated with poor prognosis. As a histone methyltransferase, the expression of EZH2 should theoretically be consistent with that of H3K27me3. However, studies have shown that the expression of EZH2 and H3K27me3 are not parallel [30, 58], and even some studies have shown that the mutually exclusive expression [59]. H3k27me3 level changes are self-sustaining, triggered by changes in transcriptional activity itself [60, 61]. However, via collecting clinical samples and analyzing them, Chen et al. ‘s study found that low expression of H3K27me3 was a powerful and independent predictor of poor differentiation of NSCLC cells and shortening of cancer-specific survival, and high expression of H3K27me3 was associated with prolongation of overall survival of NSCLC and low local recurrence and metastasis [30, 62]. Therefore, in this study, we started with a small molecule inhibitor of histone H3K27 demethylase and focused on analyzing the role of the histone demethylase inhibitor GSK-J4 in NSCLC, hoping to find new options and develop new therapeutic targets for the treatment of NSCLC.

GSK-J4 is a small molecule inhibitor of histone demethylase JMJD3/UTX, and studies have shown that JMJD3 has been identified as a potential therapeutic target to affect the invasion and migration of metastatic lung cancer cell lines [63]. GSK-J4 also showed antitumor effects in prostate cancer, breast cancer, thyroid cancer and lung cancer. Shan’s team pointed out that GSK-J4 can eliminate the expression of oncogenes induced by transcription factor specific protein 1 (SP1), thereby playing an anti-tumor role [64]. GSK-J4 can inhibit the proliferation of tumor cells, promote apoptosis, induce cell cycle arrest and inhibit epithelial mesenchymal transformation by regulating the PI3K/AKT/NF-κB signaling pathway or reducing the expression of DNA double-strand break repair genes and DNA accessibility, thus exerting anti-tumor effect [65–69]. GSK-J4 can induce metabolic reprogramming and oxidative stress in KRAS mutant lung adenocarcinoma cells to play an antitumor role [32]. GSK-J4 can also enhance chemotherapy sensitivity and radiotherapy sensitization [70, 71]. In this study, we explored the role of GSK-J4 in NSCLC EMT and verified that GSK-J4 inhibits downstream MMPs, Birc5, and CyclinD1 expression through the classical Wnt/β-catenin signaling pathway. The classic Wnt/β-catenin signaling pathway is known to play an important role in both cancer and cancer diseases and is an attractive target for treatment of different diseases. The Wnt/β-catenin signaling pathway plays a key role in different aspects of tumors, including cancer stem cells, cancer metastasis, cancer metabolism, and cancer immunity [72]. The classical Wnt pathway is usually highly conserved and activated by an autocrine/paracrine approach that binds extracellular wnt ligands to membrane receptors. Once activated, the typical Wnt pathway induces β-catenin stability and transfers it to the nucleus, ultimately participating in cell proliferation, survival, differentiation, migration, and malignant progression [37, 73–75], affecting the transcription of downstream target genes. In this study, GSK-J4 inhibited the proliferation, invasion and migration of A549 and H1299 cells, as well as EMT by blocking β-catenin and inhibiting the expression of MMPs, Birc5 and CyclinD1 downstream. When we activated the Wnt/β-catenin signaling pathway with Licl, we partially reversed the effect of GSK-J4. Therefore, we speculate that GSK-J4 may inhibit the development of NSCLC by inhibiting this signaling pathway. At the same time, due to the participation of GSK-J4 in the regulation of RNA, platinum resistance will appear in NSCLC after a period of time. Therefore, we applied GSK-J4 in combination with CDDP and found that GSK-J4 and CDDP had a synergistic effect in inhibiting NSCLC. At present, some inhibitors involving histone demethylases are undergoing preclinical research and clinical trials, opening up new directions for clinical treatment.

However, there are some limitations and deficiencies in this study. First, in our extensive literature review, we are concerned that the link between histone methyltransferase and histone demethyltransferase and H3K27me1/2/3 is not fully understood. Although EZH2 and JMJD3/UTX are theoretically opposable pairs, the regulation of H3K27 methylation is still controversial, which needs further study. Second, we combined GSK-J4 and CDDP and observed that the combination was better than the single drug, but it was not compared with the clinical standard treatment regimen. Finally, due to the limitations of clinical sample acquisition in NSCLC, current basic studies cannot use patient clinical data to verify our hypothesis. In the future, we can consider applying some emerging science such as organoids to verify it, hoping to bring new ideas to the treatment field of NSCLC.

In conclusion, the histone demethylase inhibitor GSK-J4 can inhibit the proliferation, invasion, migration and epithelial mesenchymal transformation of TGFβ1-induced NSCLC cells through Wnt/β-catenin signaling pathway, and the combination of GSK-J4 and cisplatin has a synergistic effect on the treatment of NSCLC cells. Based on this study, new prognostic factors and therapeutic targets can be identified that could help improve diagnosis and prognosis in patients with NSCLC.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Dandan Xu and Menghan Wang: Research design, cell and animal experiments. Mingyuan Wu and Yao Yu: Data statistics and analysis. Xingxuan Chen: Data collection. Danting Shen and Zhe Wen: Manuscript review. Tao Liu: Article format, grammar modification. Hong Chen: Article revision. All authors contributed to the article and approved the submission.

Funding

The research was funded and supported by the cultivation project of Heilongjiang Natural Science Foundation Joint Fund. Project Number: PL2024H120.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Patient consent for publication

Not applicable.

Ethics declarations

This animal study was reviewed and approved by the Medical Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (YJSDW2023-023).