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% CO 2 .
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 CO 2 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 1×10 4 cells/well in 100 µl DMEM, the plate was then placed in a CO 2 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 A 450 -blank group A 450 )/(cell control A 450 -blank group A 450 )] ×100%. The IC 50 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 3×10 5 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 1×10 5 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 Ca 2+ 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 IC 50 of NFZ in H1299 cells at 24 h was calculated by GraphPad Prism 8.0. The IC 50 in H1299 cells at 24 h was 19.78 µM; therefore, 20 µM was chosen as the experimental concentration.
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 ).
NFZ increases ROS and Ca 2+ 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 Ca 2+ levels ( Fig. 3C and D ) was observed.
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.
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 ).
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, Ca 2+ homeostasis in the ER is unbalanced and Ca 2+ 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 Ca 2+ induced by NFZ ( Fig. 7 ), indicating that Ca 2+ 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 ).
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 ).
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 Ca 2+ pathway ( 35 , 40 ). Under ERS, Ca 2+ is released from the ER into the cytoplasm, which causes internal Ca 2+ overload and further aggravates ROS production, thus enhancing oxidative stress. The present study demonstrated that intracellular Ca 2+ increased after NFZ treatment of H1299 cells for 24 h, and the PERK inhibitor, GSK2606414, significantly reduced the intracellular ROS, Ca 2+ 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.
Acknowledgements
Not applicable.
Glossary
Abbreviations
nifuroxazide
endoplasmic reticulum stress
protein kinase R-like ER kinase
activating transcription factor 4
DNA damage inducible transcript 3
dimethyl sulfoxide
reactive oxygen species
Janus kinase 2
signal transducer and activator of transcription 3
Funding Statement
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.
References
1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–249. doi: 10.3322/caac.21660.
2. Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. Non-small cell lung cancer: Epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc. 2008;83:584–594. doi: 10.4065/83.5.584.
3. Basumallik N, Agarwal M. StatPearls [Internet] StatPearls Publishing; Treasure Island, FL: 2022. Small cell lung cancer.
4. Jurisic V, Vukovic V, Obradovic J, Gulyaeva LF, Kushlinskii NE, Djordjević N. EGFR polymorphism and survival of NSCLC patients treated with TKIs: A systematic review and meta-analysis. J Oncol. 2020;2020:1973241. doi: 10.1155/2020/1973241.
5. Jurišić V, Obradovic J, Pavlović S, Djordjevic N. Epidermal growth factor receptor gene in non-small-cell lung cancer: The importance of promoter polymorphism investigation. Anal Cell Pathol (Amst) 2018;2018:6192187. doi: 10.1155/2018/6192187.
6. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007;7:169–181. doi: 10.1038/nrc2088.
7. Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C, et al. Drug repurposing: Progress, challenges and recommendations. Nat Rev Drug Discov. 2019;18:41–58. doi: 10.1038/nrd.2018.168.
8. Ward WC, Dodd MC. A comparative study of the in vitro bacteriostatic action of some simple derivatives of furan, thiophene, and pyrrole. J Bacteriol. 1948;56:649–652. doi: 10.1128/jb.56.5.649-652.1948.
9. CarronMCE, corp-author. Antibacterial Nitrofurfuryldene Derivatives and Methods of Using Same. 1966 December 6; US Patent US3290213A, Filed July 9 1975; issued.
10. Masunari A, Tavares LC. A new class of nifuroxazide analogues: Synthesis of 5-nitrothiophene derivatives with antimicrobial activity against multidrug-resistant Staphylococcus aureus. Bioorg Med Chem. 2007;15:4229–4236. doi: 10.1016/j.bmc.2007.03.068.
11. Said E, Zaitone SA, Eldosoky M, Elsherbiny NM. Nifuroxazide, a STAT3 inhibitor, mitigates inflammatory burden and protects against diabetes-induced nephropathy in rats. Chem Biol Interact. 2018;281:111–120. doi: 10.1016/j.cbi.2017.12.030.
12. Zhao T, Jia H, Cheng Q, Xiao Y, Li M, Ren W, Li C, Feng Y, Feng Z, Wang H, Zheng J. Nifuroxazide prompts antitumor immune response of TCL-loaded DC in mice with orthotopically-implanted hepatocarcinoma. Oncol Rep. 2017;37:3405–3414. doi: 10.3892/or.2017.5629.
13. Gan C, Zhang Q, Liu H, Wang G, Wang L, Li Y, Tan Z, Yin W, Yao Y, Xie Y, et al. Nifuroxazide ameliorates pulmonary fibrosis by blocking myofibroblast genesis: A drug repurposing study. Respir Res. 2022;23:32. doi: 10.1186/s12931-022-01946-6.
14. Saber S, Nasr M, Kaddah MMY, Mostafa-Hedeab G, Cavalu S, Mourad AAE, Gaafar AGA, Zaghlool SS, Saleh S, Hafez MM, et al. Nifuroxazide-loaded cubosomes exhibit an advancement in pulmonary delivery and attenuate bleomycin-induced lung fibrosis by regulating the STAT3 and NF-κB signaling: A new challenge for unmet therapeutic needs. Biomed Pharmacother. 2022;148:112731. doi: 10.1016/j.biopha.2022.112731.
15. Liu JY, Zhang YC, Song LN, Zhang L, Yang FY, Zhu XR, Cheng ZQ, Cao X, Yang JK. Nifuroxazide ameliorates lipid and glucose metabolism in palmitate-induced HepG2 cells. RSC Adv. 2019;9:39394–39404. doi: 10.1039/C9RA06527J.
16. Caballano-Infantes E, Terron-Bautista J, Beltrán-Povea A, Cahuana GM, Soria B, Nabil H, Bedoya FJ, Tejedo JR. Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells. World J Stem Cells. 2017;9:26–36. doi: 10.4252/wjsc.v9.i2.26.
17. Cao S, Tang J, Huang Y, Li G, Li Z, Cai W, Yuan Y, Liu J, Huang X, Zhang H. The road of solid tumor survival: From drug-induced endoplasmic reticulum stress to drug resistance. Front Mol Biosci. 2021;8:620514. doi: 10.3389/fmolb.2021.620514.
18. da Costa MOL, Pavani TFA, Lima AN, Scott AL, Ramos DFV, Lazarini M, Rando DGG. Nifuroxazide as JAK2 inhibitor: A binding mode proposal and Hel cell proliferation assay. Eur J Pharm Sci. 2021;162:105822. doi: 10.1016/j.ejps.2021.105822.
19. Luo Y, Zeng A, Fang A, Song L, Fan C, Zeng C, Ye T, Chen H, Tu C, Xie Y. Nifuroxazide induces apoptosis, inhibits cell migration and invasion in osteosarcoma. Invest New Drugs. 2019;37:1006–1013. doi: 10.1007/s10637-019-00724-4.
20. Nelson EA, Walker SR, Kepich A, Gashin LB, Hideshima T, Ikeda H, Chauhan D, Anderson KC, Frank DA. Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3. Blood. 2008;112:5095–5102. doi: 10.1182/blood-2007-12-129718.
21. Yang F, Hu M, Lei Q, Xia Y, Zhu Y, Song X, Li Y, Jie H, Liu C, Xiong Y, et al. Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model. Cell Death Dis. 2015;6:e1701. doi: 10.1038/cddis.2015.63.
22. Ye TH, Yang FF, Zhu YX, Li YL, Lei Q, Song XJ, Xia Y, Xiong Y, Zhang LD, Wang NY, et al. Inhibition of Stat3 signaling pathway by nifuroxazide improves antitumor immunity and impairs colorectal carcinoma metastasis. Cell Death Dis. 2017;8:e2534. doi: 10.1038/cddis.2016.452.
23. El-Sherbiny M, El-Sayed RM, Helal MA, Ibrahiem AT, Elmahdi HS, Eladl MA, Bilay SE, Alshahrani AM, Tawfik MK, Hamed ZE, et al. Nifuroxazide mitigates angiogenesis in Ehlrich's solid carcinoma: Molecular docking, bioinformatic and experimental studies on inhibition of Il-6/Jak2/Stat3 signaling. Molecules. 2021;26:6858. doi: 10.3390/molecules26226858.
24. Zhu Y, Ye T, Yu X, Lei Q, Yang F, Xia Y, Song X, Liu L, Deng H, Gao T, et al. Nifuroxazide exerts potent anti-tumor and anti-metastasis activity in melanoma. Sci Rep. 2016;6:20253. doi: 10.1038/srep20253.
25. Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol. 2014;24:R453–R462. doi: 10.1016/j.cub.2014.03.034.
26. Sies H. Oxidative stress: Oxidants and antioxidants. Exp Physiol. 1997;82:291–295. doi: 10.1113/expphysiol.1997.sp004024.
27. Liu L, Sun X, Guo Y, Ge K. Evodiamine induces ROS-Dependent cytotoxicity in human gastric cancer cells via TRPV1/Ca2+ pathway. Chem Biol Interact. 2022;351:109756. doi: 10.1016/j.cbi.2021.109756.
28. Sun Y, St Clair DK, Xu Y, Crooks PA, St Clair WH. A NADPH oxidase-dependent redox signaling pathway mediates the selective radiosensitization effect of parthenolide in prostate cancer cells. Cancer Res. 2010;70:2880–2890. doi: 10.1158/0008-5472.CAN-09-4572.
29. Xiao B, Liu C, Liu BT, Zhang X, Liu RR, Zhang XW. TTF1-NPs Induce ERS-Mediated apoptosis and inhibit human hepatoma cell growth in vitro and in vivo. Oncol Res. 2016;23:311–320. doi: 10.3727/096504016X14567549091341.
30. Limia CM, Sauzay C, Urra H, Hetz C, Chevet E, Avril T. Emerging roles of the endoplasmic reticulum associated unfolded protein response in cancer cell migration and invasion. Cancers (Basel) 2019;11:631. doi: 10.3390/cancers11050631.
31. Oakes SA. Endoplasmic reticulum stress signaling in cancer cells. Am J Pathol. 2020;190:934–946. doi: 10.1016/j.ajpath.2020.01.010.
32. Rozpedek W, Pytel D, Mucha B, Leszczynska H, Diehl JA, Majsterek I. The role of the PERK/eIF2α/ATF4/CHOP signaling pathway in tumor progression during endoplasmic reticulum stress. Curr Mol Med. 2016;16:533–544. doi: 10.2174/1566524016666160523143937.
33. Chen D, Fan Z, Rauh M, Buchfelder M, Eyupoglu IY, Savaskan N. ATF4 promotes angiogenesis and neuronal cell death and confers ferroptosis in a xCT-dependent manner. Oncogene. 2017;36:5593–5608. doi: 10.1038/onc.2017.146.
34. Kania E, Pająk B, Orzechowski A. Calcium homeostasis and ER stress in control of autophagy in cancer cells. Biomed Res Int. 2015;2015:352794. doi: 10.1155/2015/352794.
35. Oyadomri S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004;11:381–389. doi: 10.1038/sj.cdd.4401373.
36. Guo FJ, Liu Y, Zhou J, Luo S, Zhao W, Li X, Liu C. XBP1S protects cells from ER stress-induced apoptosis through Erk1/2 signaling pathway involving CHOP. Histochem Cell Biol. 2012;138:447–460. doi: 10.1007/s00418-012-0967-7.
37. Colovic N, Jurisic V, Terzic T, Atkinson HD, Colovic M. Immunochemotherapy for Bcl-2 and MUM-negative aggressive primary cutaneous B-cell non-Hodgkin's lymphoma. Arch Dermatol Res. 2009;301:689–692. doi: 10.1007/s00403-009-0967-1.
38. Popović B, Jekić B, Novaković I, Luković LJ, Tepavcević Z, Jurisić V, Vukadinović M, Milasin J. Bcl-2 expression in oral squamous cell carcinoma. Ann N Y Acad Sci. 2007;1095:19–25. doi: 10.1196/annals.1397.003.
39. Karan-Djurasevic T, Palibrk V, Zukic B, Spasovski V, Glumac I, Colovic M, Colovic N, Jurisic V, Scorilas A, Pavlovic S, Tosic N. Expression of Bcl2L12 in chronic lymphocytic leukemia patients: Association with clinical and molecular prognostic markers. Med Oncol. 2013;30:405. doi: 10.1007/s12032-012-0405-7.
40. Xiao B, Lin D, Zhang X, Zhang M, Zhang X. TTF1, in the form of nanoparticles, inhibits angiogenesis, cell migration and cell invasion in vitro and in vivo in human hepatoma through STAT3 regulation. Molecules. 2016;21:1507. doi: 10.3390/molecules21111507.
Associated Data
Data Availability Statement
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.