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Introduction

With the increasing incidence and mortality rates, cancer remains a primary public health problem. According to the GLOBOCAN 2020 global cancer analysis, the incidence of lung cancer ranks second in the world, with >2.2 million cases (11.4%) and >1.79 million deaths (18%) every year (1). Non-small cell lung cancer (NSCLC) accounts for 80–85% of cases of lung cancer (2,3). In recent years, EGFR-tyrosine kinase inhibitors (TKIs) have played a role in molecular targeted therapy of NSCLC, improving the prognosis of patients with NSCLC. Treatment with TKIs has revolutionized the overall survival time and quality of life in patients with NSCLC with EGFR mutations (4). The use of TKIs in NSCLC depends on the presence of EGFR mutations (5). Although EGFR plays an important role in the occurrence and development of lung cancer, patients with lung cancer with EGFR gene mutations account for 25% of NSCLC cases and are prone to drug resistance and high cytotoxicity (6). It is an urgent problem to find an effective anti-lung cancer drug with low toxicity; however, the elaboration of new mechanisms of action of drugs already in clinical application, or the study of new derivatives of drugs with low toxicity, can save the time and cost of drug development (7).

The discovery of the therapeutic potential of nitrofuran derivatives dates back to 1948 when it was discovered how they induced antimicrobial activity (8). Nifuroxazide (NFZ), a gastrointestinal antibiotic, was first patented in 1961 (England), 1966 (France) and 1966 (USA) by Robert & Carrie Laboratories (9). In previous years, it has been mainly used to prevent bacillary dysentery and enteritis (10). Previous studies have also shown that NFZ has anti-inflammatory, anti-infection, anti-diabetes, anti-renal fibrosis, anti-pulmonary fibrosis and antiproliferative activity, and ameliorates the lipid and glucose metabolism of HepG2 liver cancer cells (11–15).

The role of NFZS in small cell lung cancer is unclear. The purpose of the present study was to investigate the relationship between NFZ and H299 cell apoptosis, reactive oxygen species (ROS) and ER stress (ERS), and to explore whether NFZ is a PERK mechanism through one of the classical pathways of ERS.

Materials and methods

Materials and instruments

Human NSCLC cells, NCI-H1299 (National Infrastructure of Cell Line Resource), were cultured in DMEM high glucose medium (Hyclone; Cytiva) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a cell incubator at 37°C and 5% CO2.

NFZ and GSK2606414 were purchased from Selleck Chemicals. With dimethyl sulfoxide (DMSO) as the solvent, they were prepared into 50 mM storage solutions and stored at −80°C. The following reagents were also used in the present study: Antibodies against protein kinase R-like ER kinase (PERK; 1:1,000; #5683; CST Biological Reagents Co., Ltd.), P-PERK (1:1,000; #3179; CST Biological Reagents Co., Ltd.), activating transcription factor 4 (ATF4; 1:1,000; #11815; CST Biological Reagents Co., Ltd.), DNA damage inducible transcript 3 (CHOP; 1:1,000; #2895; CST Biological Reagents Co., Ltd.), Janus kinase 2 (JAK2; 1:1,000; #3230; CST Biological Reagents Co., Ltd.), P-JAK2 (1:1,000; #3771; CST Biological Reagents Co., Ltd.), signal transducer and activator of transcription 3 (STAT3; 1:1,000; #9139; CST Biological Reagents Co., Ltd.), P-STAT3 (1:1,000; #9145; CST Biological Reagents Co., Ltd.), HRP-linked Anti-Rabbit IgG (1:5,000; #7074; CST Biological Reagents Co., Ltd.), HRP-linked Anti-Mouse IgG (1:5,000; #7076; CST Biological Reagents Co., Ltd.), HRP-conjugated β-actin (1:5,000; #4967; CST Biological Reagents Co., Ltd.) and HRP-conjugated GAPDH (1:5,000; #3683; CST Biological Reagents Co., Ltd.); Annexin V/PI kit and BCA kit (both from Yeasen Biotechnology Co., Ltd.); Brite 670 (AmyJet Scientific, Inc.) and Fura Red AM (A&D Technology); DAPI (Sigma-Aldrich; Merck KgaA).

The following instruments were used in the present study: BB15 CO2 incubator (Thermo Fisher Scientific, Inc.), desktop low-temperature 5810R centrifuge, BD Accuri™ C6 flow cytometer (BD Biosciences), Olympus IX-B53 inverted fluorescence microscope (Olympus Corporation), Bio-Rad 550 enzyme reader and electrophoresis/membrane transfer apparatus (both from Bio-Rad Laboratories, Inc.), and BioSpectrum 810 Imaging System (Analytik Jena AG).

Cell Counting Kit-8 (CCK-8) cell viability assay

H1299 cells in the logarithmic growth stage were divided into two groups, the DMSO and NFZ groups. The NFZ group was divided into seven sub-groups depending on the concentration of NFZ and there were three replicates in each group. The single cell suspension was aliquoted into 96-well plates, with 1×104 cells/well in 100 µl DMEM, the plate was then placed in a CO2 cell incubator at 37°C. When the cell adhesion density reached 75%, DMEM high glucose medium containing 1% DMSO was added to the DMSO group, and DMEM high glucose medium containing 0.25, 0.5, 1, 2, 4, 8, and 16 µM NFZ was added to the NFZ sub-groups. At 24, 48 and 72 h time points, 10 µl CCK-8 solution (Yeasen Biotechnology Co., Ltd.) was added to each well and incubated in the cell incubator for 1 h, and an empty well was also filled with 10 µl CCK-8 solution as a blank group. The plate was inserted into the Bio-Rad 550 enzyme microplate reader and each group was measured at an OD of 450 nm. The experiment was repeated three times. Relative cell viability rate=[(cell experimental group A450-blank group A450)/(cell control A450-blank group A450)] ×100%. The IC50 of NFZ was calculated using GraphPad Prism 8.0 software (Dotmatics).

H1299 cells were washed with PBS and digested with 25% trypsin-EDTA to form a single cell suspension that was added to 96-well plates. The plates were randomly divided into a DMSO, NFZ, GSK2606414 and NFZ + GSK2606414 group, and each group had three wells. At 24 h, cells in each group were treated, except the cells in the DMSO group. Cells in the NFZ group were treated with 20 µM NFZ medium, and cells in the GSK2606414 group were treated with 1 µM GSK2606414 medium. Cells in the NFZ + GSK2606414 group were treated with 1 µM GSK2606414 and 1 h later, 20 µM NFZ and 1 µM GSK2606414 were added in the form of liquid exchange. All plates were then placed in the incubator for incubation at 37°C. At 24 h time points, 10 µl CCK-8 solution was added to each well and incubated in the cell incubator for 1 h. The plates were inserted into the Bio-Rad 550 enzyme microplate reader and each group was measured at an OD of 450 nm.

Optical microscopy

After digestion in 0.25% trypsin, H1299 cells were seeded into 24-well plates at 3×105 cells/ml in each well. The cells were placed in the incubator, the morphological changes were observed and images were captured under an optical microscope after 24 h.

Inverted fluorescence microscopy

The grouping and dosing methods aforementioned were repeated and, 24 h later, 10 µl Brite 670 was added to the cells and the cells were incubated at 37°C for 30 min in the dark. The cells were then washed twice with 1X PBS and images were captured using an inverted IX-B53 fluorescence microscope at ×200 magnification. ImageJ (v1.53c) was used for data analysis.

Flow cytometry

H1299 cells were washed with PBS and digested with 25% trypsin-EDTA to form a single cell suspension that was added to 10-cm dishes for culturing. The plates were randomly divided into a DMSO, NFZ, GSK2606414 and NFZ + GSK2606414 group. At 24 h, cells in each group were treated, except the cells in the DMSO group. Cells in the NFZ group were treated with 20 µM NFZ medium, and cells in the GSK2606414 group were treated with 1 µM GSK2606414 medium. Cells in the NFZ + GSK2606414 group were treated with 1 µM GSK2606414 and 1 h later, 20 µM NFZ and 1 µM GSK2606414 were added in the form of liquid exchange. All plates were then placed in the incubator for incubation at 37°C for 24 h time. After treatment, the cells were digested with 0.25% trypsin and collected by centrifugation (300 × g, 4°C, 5 min). After centrifugation, the cells were resuspended in PBS, the cell density was adjusted to 1×105 cells/ml and the cells were washed with PBS a further three times. For detection, 100 µl 1X binding buffer containing 5 µl Annexin V-FITC and 10 µl PI were added into each tube and incubated at 37°C for 60 min. Subsequently, 400 µl 1X binding buffer was added to each tube for cell resuspension, and cell apoptosis was detected by BD Accuri C6 flow cytometer and the data were analyzed using FlowJo 10.0 (FlowJo LLC).

To stain intracellular ROS, 10 µl 100 µM Brite 670 was added to the treated cells and ROS was detected by flow cytometry. Brite 670 was first incubated for 30 min, and then DAPI was added for 15 min. Finally, the double-stained cells were cleaned twice with PBS, which led to observation and photography under fluorescence microscope. In addition, 200 µl Fura Red AM was added to the treated cells for intracellular Ca2+ staining, and the emission wavelength was detected at 550 nm. The BD Accuri C6 flow cytometer was used for detection and the data were analyzed using FlowJo 10.0 (FlowJo LLC).

Western blot analysis

After the aforementioned treatments, the cells were washed with PBS and centrifuged (300 × g, 4°C, 5 min) to collect the suspended cells. The cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) containing 1:100 protease inhibitor and 1:100 phosphatase inhibitor, and a BCA kit was used to detect protein concentration.

Proteins (20 µg) were separated by SDS-PAGE on 10 or 12% gels. Electrophoresis and PVDF membrane transfer were performed using Bio-Rad electrophoresis and membrane transfer apparatus. For blocking, the membranes were incubated with 5% BSA (Biological Industries) on a 4°C with agitation for 2 h. After blocking, the membranes were washed three times for 10 min intervals. Primary antibodies were added at a dilution of 1:1,000 and incubated at 4°C overnight. Subsequently, the membranes were washed three times for 10 min intervals. After the addition of the secondary antibody at a ratio of 1:5,000, the membranes were incubated with agitation for 2 h at 4°C and then washed three times for 10 min intervals. Finally, ECL chemiluminescence developer (Yeasen Biotechnology Co., Ltd.) was added to the membranes and, after full reaction, the membranes were placed in a Biospectrum 810 Imaging system for exposure development. The protein expression bands were observed and recorded, and the gray values of each band were analyzed by ImageJ software (v1.53c).

Statistical analysis

GraphPad Prism 8.0 software was used for statistical analysis. The data are presented as the mean ± SD. Student's t-test was used for data comparison between two unpaired groups. One-way ANOVA was applied for multiple group comparisons and Dunnett's post hoc test was performed to compare the means of multiple groups with those of a single group. All experiments were repeated three times. P<0.05 was considered to indicate a statistically significant difference.

Results

NFZ inhibits the viability of H1299 cells

After H1299 cells were treated with 0.25, 0.5, 1, 2, 4, 8 and 16 µM NFZ for 24, 48 and 72 h, the viability of the H1299 cells decreased significantly compared with that in the DMSO group and showed significant cytotoxicity, which was proportional to time and dose (Fig. 1). The IC50 of NFZ in H1299 cells at 24 h was calculated by GraphPad Prism 8.0. The IC50 in H1299 cells at 24 h was 19.78 µM; therefore, 20 µM was chosen as the experimental concentration.

Figure 1. Effects of different concentrations of NFZ (0.25, 0.5, 1, 2, 4, 8 and 16 µM) on H1299 cell viability at (A) 24, (B) 48 and (C) 72 h. Relative cell viability was determined by Cell Counting Kit-8 assay. Compared with in the DMSO group, H1299 cell viability of even the low dose (0.25 µM) group was significantly decreased after 24 h of treatment. *P<0.05, **P<0.01, ***P<0.001. DMSO, dimethyl sulfoxide; NFZ, nifuroxazide.

NFZ induces the apoptosis of H1299 cells

H1299 cells were treated with 20 µm NFZ for 6, 12 and 24 h, and the degree of apoptosis of H1299 cells increased in a time-dependent manner. After 6 h, apoptosis began to increase significantly (Fig. 2).

Figure 2. Apoptosis of H1299 cells induced by NFZ. H1299 cells were treated with (Aa) DMSO for 24 h, or with 20 µM NFZ for (Ab) 6, (Ac) 12 and (Ad) 24 h. Flow cytometry was used for detection. (B) Results of flow cytometric analysis. ***P<0.001. DMSO, dimethyl sulfoxide; NFZ, nifuroxazide.

NFZ increases ROS and Ca2+ levels in H1299 cells

After exposure to 20 µM NFZ for 24 h, compared with those in the DMSO control group, an increase in the intracellular ROS (Fig. 3A and B) and Ca2+ levels (Fig. 3C and D) was observed.

Figure 3. ROS and Ca2+ levels of H1299 cells treated with NFZ. ROS levels in H1299 cells after 24 h of (Aa) DMSO or (Ab) 20 µM NFZ treatment. (B) Quantification of ROS levels. Ca2+ levels in H1299 cells after 24 h of (Ca) DMSO or (Cb) 20 µM treatment. (D) Quantification of Ca2+ levels. Data are presented as the mean ± SD. **P<0.01. DMSO, dimethyl sulfoxide; NFZ, nifuroxazide; ROS, reactive oxygen species.

NFZ induces activation of the ERS-related PERK pathway

NFZ significantly increased the expression levels of P-PERK, ATF4 and CHOP, key proteins in the PERK pathway of ERS, and unphosphorylated PERK decreased with treatment of the drug, compared with that in the DMSO control group (Fig. 4). These results were observed even after 6 h of treatment.

Figure 4. Changes in PERK, P-PERK, ATF4 and CHOP expression. (Aa) Expression of PERK, P-PERK and CHOP in H1299 cells exposed to NFZ for 6, 12 and 24 h. (Ab) The ratio of PERK expression relative to β-actin expression in each group. (Ac) The ratio of P-PERK expression relative to β-actin expression in each group. (Ad) The ratio of p-PERK/β-actin to PERK/β-actin. (Ae) The ratio of CHOP expression relative to β-actin expression in each group. (Ba) Expression of ATF4 in H1299 cells exposed to NFZ for 6, 12 and 24 h. (Bb) The ratio of ATF4 expression relative to GAPDH expression in each group. Data are presented as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001. AFT4, activating transcription factor 4; CHOP, DNA damage inducible transcript 3; DMSO, dimethyl sulfoxide; NFZ, nifuroxazide; PERK, protein kinase R-like ER kinase.

GSK2606414 reduces the morphological changes and apoptosis induced by NFZ

Compared with in the DMSO group, the cell body of H1299 cells treated with NFZ decreased and became round, and the cells did not form colonies. A small amount of granular material appeared in the cells, there were more cell fragments in the culture medium and the cell viability was significantly decreased (Fig. 5A and B). The addition of GSK2606414 reduced the morphological changes induced by NFZ. The addition of 1 µM GSK2606414 also reduced the NFZ-induced apoptosis and cytotoxicity in H1299 cells (Fig. 5B-D).

Figure 5. Effect of the protein kinase R-like ER kinase inhibitor, GSK2606414, on NFZ-induced apoptosis. Morphological changes of cells treated with NFZ for 24 h; (Aa) DMSO, (Ab) NFZ, (Ac) GSK2606414 and (Ad) NFZ + GSK2606414 groups. (B) Relative cell viability determined by Cell Counting Kit-8 assay. NFZ significantly reduced cell viability compared with the DMSO group (***P<0.001). The cytotoxicity of NFZ + GSK2606414 group was significantly lower than that of NFZ group (###P<0.01). Apoptosis of cells treated with (Ca) DMSO, (Cb) NFZ, (Cc) GSK2606414 or (Cd) NFZ + GSK2606414. (D) Results of flow cytometric analysis. NFZ significantly reduced the cell apoptosis rate compared with the DMSO group (***P<0.001). Cell apoptosis rate induced by NFZ was significantly reduced by GSK2606414 (###P<0.01). DMSO, dimethyl sulfoxide; NFZ, nifuroxazide.

GSK2606414 can inhibit ERS-induced by NFZ

GSK2606414 significantly reduced the increase of intracellular ROS induced by NFZ (Fig. 6). ROS is closely related to oxidative stress, and PERK is one of the classical pathways of ERS (16,17). The results of the present study indicated that the increase of intracellular ROS level induced by NFZ can lead to ERS. After ERS occurs, Ca2+ homeostasis in the ER is unbalanced and Ca2+ in the ER is released into the cytoplasm, which increases the level of ROS in the cytoplasm and further aggravates oxidative stress (16,17). GSK2606414 inhibited the increase in intracellular Ca2+ induced by NFZ (Fig. 7), indicating that Ca2+ release from the ER is an important mechanism of NFZ-induced H1299 cell apoptosis. GSK2606414 also inhibited the increase in P-PERK, ATF4 and CHOP protein expression levels induced by NFZ (Fig. 8).

Figure 6. Effects of protein kinase R-like ER kinase inhibitor, GSK2606414, on intracellular ROS levels. (A) Levels of ROS in DMSO, NFZ, GSK2606414 and NFZ + GKS2606414 treated cells were detected by inverted fluorescence microscopy. (B) Average fluorescence intensity in each group. ***P<0.001, ###P<0.01. Scale bar, 20 µm. DMSO, dimethyl sulfoxide; NFZ, nifuroxazide; ROS, reactive oxygen species.

Figure 7. Changes in intracellular Ca2+ levels after pre-treatment with GSK2606414. (Aa) DMSO, (Ab) NFZ, (Ac) GSK2606414 and (Ad) NFZ + GSK2606414 groups. (B) Mean fluorescence intensity in each group. ##P<0.01, ***P<0.001. DMSO, dimethyl sulfoxide; NFZ, nifuroxazide.

Figure 8. GSK2606414 inhibits the increases in P-PERK, ATF4 and CHOP protein expression induced by NFZ. (Aa) Changes in PERK, P-PERK, ATF4 and CHOP in DMSO, NFZ, GSK2606414 and NFZ + GSK2606414 groups. (Ab) The ratio of PERK expression relative to β-actin expression in each group. (Ac) The ratio of P-PERK expression relative to β-actin expression in each group. (Ad) The ratio of P-PERK/β-actin to PERK/β-actin. (Ae) The ratio of CHOP expression relative to β-actin expression in each group; (Ba) Expression of ATF4 in DMSO, NFZ, GSK2606414 and NFZ + GSK2606414 groups. (Bb) The ratio of ATF4 expression relative to GAPDH expression in each group. *P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01, ###P<0.001. AFT4, activating transcription factor 4; CHOP, DNA damage inducible transcript 3; DMSO, dimethyl sulfoxide; NFZ, nifuroxazide; PERK, protein kinase R-like ER kinase.

NFZ reduces the phosphorylation of JAK2 and STAT3

The western blotting results showed that the phosphorylation levels of STAT3 and JAK2 in the NFZ group were significantly lower than those in the DMSO group. These findings indicated that NFZ may be an inhibitor of JAK2 and STAT3 (Fig. 9).

Figure 9. Changes in JAK2 and STAT3 phosphorylation after H1299 cells were exposed to NFZ for 24 h. (Aa) Expression of P-JAK2 and JAK2 in DMSO or NFZ treated groups. (Ab) Ratio of P-JAK2/β-actin to JAK2/β-actin. (Ba) Expression of P-STAT3 and STAT3 in DMSO or NFZ treated groups. (Bb) Ratio of P-JAK2/β-actin to JAK2/β-actin. *P<0.05, **P<0.01. DMSO, dimethyl sulfoxide; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; NFZ, nifuroxazide.

Discussion

NFZ has become a research focus in recent decades due to its antitumor and JAK2 inhibitor activity (18). NFZ, an oral nitrofuran antibiotic, reduces the viability of a number of cancer cells (such as osteosarcoma, multiple myeloma, breast cancer, colorectal carcinoma and solid Ehrlich carcinoma cells) by inhibiting STAT3 phosphorylation (19–23). NFZ can significantly inhibit the migration and invasion of osteosarcoma cells through P-STAT3, MMP-2 and MMP-9 mediated signaling pathways (19). In addition, studies have shown that NFZ has antitumor cell proliferation and metastasis effects on liver cancer, breast cancer, multiple myeloma and melanoma (12,20–22,24). The occurrence of malignant tumors is due to inhibition of the apoptosis mechanism, leaving cells unable to carry out cell death and cell elimination. The induction of tumor cell apoptosis is the main way to eliminate cancer cells. Both the ER and mitochondrial pathways are typical apoptotic pathways. A previous study reported that NFZ can induce the apoptosis of osteosarcoma cells through the mitochondrial pathway (19).

Oxidative stress refers to imbalance of the redox system and the significant increase of free radicals, which exceeds the scavenging capacity of the endogenous antioxidant system (25). However, when the production of free radicals exceeds the scavenging capacity of the body, excessive ROS will accumulate in the cells and attack biological macromolecules and organelles, resulting in different degrees of oxidative stress response to DNA, proteins, lipids and carbohydrates (26). Certain studies have also demonstrated that ROS in tumor cells exceed the tolerance capacity of cells, resulting in changes in tumor cell apoptosis (27,28). The present study demonstrated that intracellular ROS were significantly increased in H1299 lung cancer cells treated with NFZ for 24 h. CCK-8 and flow cytometry experiments also demonstrated that NFZ may induce the apoptosis of H1299 cells via oxidative stress. ERS is closely related to oxidative stress and early ERS plays a cytoprotective role, but sustained ERS activates the apoptosis signaling pathway to protect the functional stability of cells (29). PERK, a member of the elF2a protein kinase family, is also a type I transmembrane protein kinase located in the ER membrane, which mainly transduces ERS signals via three typical ERS-induced apoptotic pathways involving inositol-requiring enzyme 1, PERK and ATF6 (30,31). PERK is also activated during tumor progression (32).

ERS can promote apoptosis mainly by promoting the expression of transcription factors, such as ATF4 and CHOP. ATF4 plays a key role in the transcriptional regulation of pro-survival genes mainly related to oxidative stress, autophagy, protein folding, amino acid synthesis and cell differentiation (33). The PERK signaling pathway not only reduces the folding pressure of newly synthesized proteins from the ER, but also specifically enhances the transcription level of certain genes through the fine regulatory mechanism. The upregulation of these genes is mediated by the transcription factor, ATF4 (32,34). After synthesis, ATF4 is translocated to the nucleus, and acts as a transcription factor to upregulate the transcriptional expression of molecular chaperones in the ER and amino acid transport proteins. However, sustained overexpression of ATF4 promotes the upregulation of the gene encoding CHOP (35,36). It has been confirmed that ERS in tumor cells leads to the activation of CHOP (32,34). Apoptosis is primarily inhibited by the presence of the anti-apoptotic molecule, Bcl-2, which has previously been observed in various tumor types (including primary cutaneous B-cell non-Hodgkin's lymphoma cells and oral squamous cells) and high expression of Bcl-2 is associated with survival and therapeutic response (37,38). A previous study reported that NFZ induces apoptosis in breast cancer through the activation of cleaved caspase-3 and Bax and the downregulation of Bcl-2 (21). Similarly, in another study, NFZ upregulated CHOP protein levels and induced the caspase molecular cascade reaction by reducing Bcl-2 levels, thus leading to apoptosis of tumor cells (39). In the present study, the experimental results demonstrated that NFZ significantly increased the expression levels of P-PERK, ATF4 and CHOP. These results indicated that NFZ may induce H1299 cell apoptosis through the PERK ERS pathway. ERS is an important pathway for cell apoptosis, and its process requires the participation of a number of molecular chaperones and specific proteins, including the transcriptional activation of CHOP and the Ca2+ pathway (35,40). Under ERS, Ca2+ is released from the ER into the cytoplasm, which causes internal Ca2+ overload and further aggravates ROS production, thus enhancing oxidative stress. The present study demonstrated that intracellular Ca2+ increased after NFZ treatment of H1299 cells for 24 h, and the PERK inhibitor, GSK2606414, significantly reduced the intracellular ROS, Ca2+ levels and the protein expression levels of P-PERK, ATF4 and CHOP, thus inhibiting the level of apoptosis.

As depicted in Fig. 10, NFZ may induce apoptosis of human NSCLC H1299 cells by activating the PERK-ATF4-CHOP pathway of ERS. The present study demonstrated the molecular mechanism of NFZ cytotoxicity from the perspective of ERS-mediated apoptosis, which may provide a new basis for revealing the cytotoxicity of NFZ to tumor cells. The present study also provided some toxicological data to support the scientific and rational use of NFZ and provides a new option and molecular biological basis for the treatment of NSCLC. However, the in vitro culture cannot completely simulate the in vivo internal environment. Therefore, in vivo experiments will be conducted in a future study.

Figure 10. As a classical inhibitor of JAK2/STAT3, NFZ can induce ERS by increasing the intracellular ROS levels, thereby releasing Ca2+ from the ER into the cytoplasm, increasing the cytoplasmic Ca2+ levels and further increasing the intracellular ROS levels. In addition, the PERK pathway, the classical pathway of ERS, is activated, and the level of CHOP in the nucleus is increased, thus inducing the apoptosis of H1299 human non-small cell lung cancer cells. AFT4, activating transcription factor 4; CHOP, DNA damage inducible transcript 3; ERS, ER stress; JAK2, Janus kinase 2; NFZ, nifuroxazide; PERK, protein kinase R-like ER kinase; ROS, reactive oxygen species; STAT3, signal transducer and activator of transcription 3.

Acknowledgements

Not applicable.

Funding

Funding was provided by The Qingdao Applied Basic Research Program (grant no. 19-6-2-31-cg).

Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available as the subject has not been fully concluded and the data cannot be disclosed for the time being, but are available from the corresponding author on reasonable request.

Authors' contributions

DL and LL co-wrote the manuscript. In addition, DL and LL jointly performed experiments, analyzed the experimental data and drew the pathway map. DL, LL and FL searched the literature together. FL corrected the language for some important content and helped to design some of the experiments. KG and CM designed the study together and revised the article. FL also helped to design the study. KG gave the funding support. All authors read and approved the final manuscript. All authors confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References