NSCLC cancer cell lines H1299, PC-9, PC-9-IR and H1975, and the colorectal cancer cell line Caco2 were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. All cells were cultured in RPMI 1640 medium (Nanjing BioChannel Biotechnology Co., Ltd.) containing 10% heat-inactivated fetal bovine serum (GeminiBio) at 37°C in an incubator containing 5% CO₂.

Lipofectamine^® RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect small interfering RNA (siRNA) into cells. According to the manufacturer's protocol, H1975, PC-9 and PC-9IR cells at 70% confluence were used for transfection. Briefly, 1 pmol siRNA/well was used for the transfection of cells on a 96-well plate, and 30 pmol siRNA/well was used for the transfection of cells on a 6-well plate. After a 5-min incubation at room temperature, the RNAiMAX-siRNA mixture was added to either 6-well or 96-well plates. The cells were incubated for 48 h without media replacement, after which subsequent experiments were performed. The 96-well plates were used for MTT and cell proliferation assays. The 6-well plates were used for the analysis of transcript or protein expression levels. The siRNA sequences used in the present study are listed in Table I. Negative control siRNA and siBORIS were synthesized by Xiangyin Biotechnology Co., Ltd. The cells were then incubated at 37°C in an incubator containing 5% CO₂. After 48 h, the cells were used for subsequent experiments. Following transfection, cells were treated with their respective drug treatments, with the control group receiving an equal volume of DMSO. Atractyloside (cat. no. HY-N1462), the TKI inhibitor gefitinib (cat. no. HY-50895) and erlotinib (cat. no. HY-50896) were purchased from MedChemExpress. Cells were treated with gefitinib (50 µM) 4 h post-transfection at room temperature. Cells were subjected to experiments after 48 or 96 h of gefitinib treatment. In addition, cells were treated with gefitinib (1 µM), erlotinib (5 µM), or atractyloside (1, 2.5 or 5 µM) for 48 h at 37°C prior to performing Cell Counting Kit (CCK)-8 assays, western blotting and reverse transcription-quantitative PCR (RT-qPCR).

A total of 3,000 cells/well were seeded in a 96-well plate for transfection or drug treatment. Subsequently, MTT (500 µg/ml; cat. no. M2128; Sigma-Aldrich; Merck KGaA) was added to the cells and incubated for 4 h at 37°C, and 100 µl dimethyl sulfoxide was added for 15 min at room temperature. Signals were recorded using a BioTek Synergy 2 plate reader at a wavelength of 490 nm (BioTek; Agilent Technologies, Inc.).

A total of 3,000 cells/well were seeded in a 96-well plate and underwent drug treatment. After treatment with drugs (gefitinib, 1 µM; erlotinib, 5 µM; atractyloside, 5 µM) for 48 h at 37°C, the cell culture medium was discarded, and 100 µl medium containing 10 µl CCK-8 (cat. no. K1018; APeXBIO Technology LLC) reagent was added. The cells were then incubated for 1 h at 37°C and signals were recorded using a BioTek Synergy 2 plate reader at a wavelength of 450 nm.

H1299, Caco2, PC-9 and H1975 cells were cultured in a 6-well plate and were lysed using RIPA buffer (cat. no. 20188; MilliporeSigma) containing PMSF (1:100; cat. no. ST506; Beyotime Institute of Biotechnology) and Roche cOmplete™ Protease Inhibitor Cocktail (1:25; cat. no. 04693116001; Sigma-Aldrich; Merck KGaA). After centrifugation at 12,000 × g for 30 min at 4°C, the supernatants were collected, and the total protein was quantified using a detergent-compatible Bradford protein assay kit (cat. no. P0006C; Beyotime Institute of Biotechnology). Samples (30 µg/lane) were separated by SDS-PAGE on a 10% gel and were transferred onto a PVDF membrane (cat. no. ISEQ00010-PVDF; MilliporeSigma). The membrane was blocked with a protein-free rapid blocking buffer (cat. no. PS108P; New Cell & Molecular Biotech Co., Ltd.) for 15 min at room temperature and then incubated at 4°C overnight with the following antibodies: Mouse anti-GAPDH (1:500,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.), rabbit anti-XRCC4 (1:1,000; cat. no. 15817-1-AP; Proteintech Group, Inc.), mouse anti-BORIS (1:1,000; cat. no. sc-377085; Santa Cruz Biotechnology, Inc), rabbit anti-AKT (1:1,000; cat. no. 9272; Cell Signaling Technology, Inc.) and mouse anti-phosphorylated (p)-AKT (1:1,000; cat. no. 66444-1-Ig; Proteintech Group, Inc.). After washing with TBS-1% Tween (TBST) three times (10 min/wash), the membrane was incubated with HRP-conjugated secondary antibodies (anti-rabbit and anti-mouse; 1:5,000; cat. nos. DW-GAR007 and DW0990-100; Hangzhou Dawen Biological Co., Ltd.) for 2 h at room temperature. Signals were detected after washing with TBST three times (10 min/wash) using the Ultrasensitive ECL Kit (cat no. P2300; New Cell & Molecular Biotech Co., Ltd)and ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc.). The relative expression of the protein bands was semi-quantified using ImageJ version 1.53 software (National Institutes of Health).

The RNA of treated cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) through ethanol precipitation. cDNA was reverse transcribed using the Hifair^® II 1st Strand cDNA Synthesis Kit (gDNA digester plus) (cat. no. 11121ES60; Shanghai Yeasen Biotechnology Co., Ltd.), and was used for qPCR analysis. For RT, the temperature settings were as follows: 25°C for 5 min, 42°C for 30 min and 85°C for 5 min. qPCR was performed using the 2X T5 Fast qPCR Mix (SYBR Green; cat. no. 11201ES08; Shanghai Yeasen Biotechnology Co., Ltd.) and a CFX connect real-time PCR detection system (Bio-Rad Laboratories, Inc.). According to the manufacturer's protocol, the thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 30 sec. GAPDH was used as an internal reference for normalization. The primers used for qPCR are listed in Table II. The qPCR results were analyzed using the 2^−ΔΔCq method (19).

BORIS expression in NSCLC was determined using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). The Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) datasets GSE19188 (20) and GSE63074 (21) were utilized for bioinformatics analysis.

GraphPad Prism 8 software (Dotmatics) was used for all statistical analyses. All experiments were performed in triplicate. Data are presented as the mean ± standard deviation. Statistical differences were calculated using one-way or two-way ANOVA followed by Tukey's multiple comparisons test, paired Student's t-test or unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Bioinformatics analysis was performed using the R2 Genomics Analysis and Visualization Platform. Based on the GSE19188 dataset, BORIS expression was elevated in primary NSCLC tissues compared with in normal lung tissues (Fig. 1A). From the GSE63074 dataset, survival rate data were used to perform a risk stratification analysis, categorizing patients into high-risk and low-risk groups according to a previously described method (22); the analysis indicated a significant upregulation of BORIS expression in the high-risk NSCLC group (Fig. 1B). Given that EGFR upregulation or mutation was prevalent in high-risk NSCLC cases, further investigation into the potential association between BORIS and EGFR expression/mutation is warranted.

The H1975 cell line comprises NSCLC cells resistant to gefitinib due to the T790M mutation. The present study knocked down BORIS in H1975 cells and the results demonstrated that the cell viability was significantly decreased in response to successful transfection with siBORIS (Fig. 1D). This finding indicated that the presence of BORIS may maintain the stability of H1975 cells and that its knockdown could be beneficial for treating drug-resistant lung cancer.

To further explore the function of BORIS in TKI resistance, PC-9, PC-9IR and H1975 cells underwent BORIS knockdown and gefitinib treatment. PC-9 is an EGFR wild-type NSCLC cell line, whereas PC-9IR and H1975 are EGFR-mutant cells that are resistant to TKIs.

siBORIS effectively reduced the viability of NSCLC cells (Fig. 1D-F). All cell transfections were successful. When siBORIS was combined with gefitinib treatment, cell viability was significantly reduced compared with gefitinib treatment alone, with a more obvious effect observed on the drug-resistant cell lines H1975 and PC9-IR (Fig. 1D and 1F). Verification of the knockdown efficiency of siBORIS is presented in Fig. 1G. These findings suggested that the knockdown of BORIS may be beneficial for lung cancer resistance. Western blot analysis confirmed that the expression of BORIS was decreased in response to siBORIS.

In a previous study, a drug that could mimic BORIS knockdown was identified (23). To identify bioactive drugs that might mimic the effects of BORIS knockdown, genes regulated in BORIS-silenced Caco2 cells were analyzed using microarray and a connectivity map database was screened for associated drugs in our previous study (23). Based on gene expression patterns and drug correlation analysis, metronidazole and atractyloside were identified as promising candidates for further study (23). These previous findings using the Caco2 cell line demonstrated that atractyloside (Fig. 1C) inhibited cell viability (23).

The H1299 (wild-type EGFR) and H1975 (EGFR mutation) NSCLC cells were used for assessing the response of siBORIS or atractyloside treatment. The results demonstrated that siBORIS treatment downregulated the expression of DNA repair-related genes, including BRCA-1, MSH6 and c-myc (Fig. 2B). Atractyloside treatment, on the other hand, increased XRCC4 expression while downregulating BRCA-1 (Fig. 2D), which is consistent with our previous observations (23). In H1299 and H1975 cells, atractyloside treatment resulted in DNA damage and upregulation of XRCC4 expression (Fig. 2B and D). These results indicated that atractyloside could mimic the effects of siBORIS to regulate the downstream genes (Fig. 2C and D); however, atractyloside treatment did not influence BORIS expression (Fig. 2A). In the present study atractyloside was used instead of siBORIS in subsequent experiments to regulate BORIS-related downstream genes.

To avoid using a high concentration of atractyloside, which would induce mitochondrial permeability transition and cause apoptosis (4), 5 µM atractyloside was selected for application in two lung cancer cell lines, the wild-type cell line PC-9 and the T790M mutant drug-resistant cell line H1975.

In PC-9 cells, it was observed that, after 2 days of administration, gefitinib inhibited cell proliferation, and after 4 days administration, the proliferation of PC-9 cells was significantly inhibited by gefitinib (Fig. 3A). Atractyloside was shown to suppress PC-9 cell proliferation, but was less effective than gefitinib. In addition, the effect of the two-drug combination on cell proliferation was not significant, thus indicating that atractyloside had little effect on wild-type lung cancer cells. In H1975 cells, gefitinib at a concentration of 1 µM did not affect cell proliferation (Fig. 3B). However, when used in combination with atractyloside, cell proliferation was significantly decreased, indicating that the combined administration of atractyloside and gefitinib may affect the proliferation of TKI-resistant cells.

To further verify the inhibitory effect of combination therapy on EGFR-mutant cells, the 2nd-generation EGFR-TKI inhibitor erlotinib was used. After treatment with the drugs for 96 h, a combination of erlotinib and atractyloside suppressed H1975 cell proliferation better than erlotinib alone (Fig. 3C). Atractyloside demonstrated cytotoxic effects on cancer cells, with increasing concentrations inhibiting H1975 cell proliferation (Fig. 3D). When combined with gefitinib, the effect of atractyloside on drug-resistant lung cancer cells was stronger than that on wild-type cells, indicating that the BORIS pathway may be associated with lung cancer resistance. Western blot analysis indicated that atractyloside may suppress NSCLC cell proliferation by inhibiting AKT phosphorylation (Fig. 3E and F). This finding aligns with the results of a previous study demonstrating that AKT phosphorylation promotes lung cancer cell proliferation (24). AKT is a downstream factor of EGFR and can be used to examine the severity of cancer; therefore, the inhibition of AKT phosphorylation indicated that EGFR-related signaling was suppressed by atractyloside.

After knockdown of the expression of BORIS in the H1975 cell line, a decrease in the expression levels of the homologous recombination-related genes c-myc, BRCA-1 and the mismatch repair gene MSH6 (25), was detected (Fig. 2C). These findings were consistent with our previous results (12) and indicated the existence of BORIS-stabilized cell DNA. To further verify the effects of atractyloside on H1975 and PC-9 cells, XRCC4 protein expression was detected. The data demonstrated a consistent trend of XRCC4 upregulation, observed in response to both BORIS knockdown and atractyloside treatment (Fig. 4A-D). In PC-9 cells, BORIS knockdown elevated the expression of XRCC4 (Fig. 4A), as did atractyloside (Fig. 4B). In addition, in H1975 cells, BORIS knockdown elevated the expression of XRCC4 (Fig. 4C), as did atractyloside (Fig. 4D). In summary, atractyloside may disrupt DNA stability in H1975 cells and PC-9 cells.