Forchlorfenuron exposure Induces hepatocyte Apoptosis via MKK3/p38/ATF-2 signaling Pathway

doi:10.21203/rs.3.rs-4212817/v1

Download PDF

Article

Forchlorfenuron exposure Induces hepatocyte Apoptosis via MKK3/p38/ATF-2 signaling Pathway

https://doi.org/10.21203/rs.3.rs-4212817/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Forchlorfenuron is a widely used plant cytokinin in traditional Chinese medicine and agricultural cultivation to boost resistance, postpone senescence, and increase productivity. However, improper forchlorfenuron use results in excessive residues and contamination, raising health and safety concerns. The in vitro toxicity of forchlorfenuron on HepaRG cells was investigated in our research. Results showed that forchlorfenuron inhibited HepaRG cell viabilities in a concentration and time-dependent manner. Forchlorfenuron induced cellular apoptosis and the increased intracellular reactive oxygen species (ROS) indicated the participation of oxidative stress. Molecular docking and network pharmacology data suggested that the hepatotoxicity of forchlorfenuron might involve the MAPK signaling pathway. After 24 hours of forchlorfenuron exposure, the p38-MAP kinase, upstream kinases MKK3, and the transcription factor ATF-2 was maximally activated. Apoptosis induced by forchlorfenuron was significantly reduced by pretreatment of the P38 inhibitor SB203580. These findings implicated that HepaRG hepatocyte injuries were generated by forchlorfenuron through the induction of cellular apoptosis via MKK3/p38/ATF-2 pathways. Forchlorfenuron application should be closely managed to prevent potential liver damage.

Biological sciences/Cell biology/Cell death

Health sciences/Risk factors

Forchlorfenuron

hepatocyte apoptosis

network pharmacology

molecular docking

MKK3/p38/ATF-2

Plant growth regulator (PGR) refers to a wide class of synthetic compounds that have phytohormone properties and are frequently employed in agriculture to boost fruit and plant yield, enhance flavor, and accelerate maturation. In recent years, common Chinese herbal medicine have additionally been produced using PGR to meet the growing demand for Traditional Chinese Medicine (TCM). However, improper PGR application might worsen safety problems as well as quality issues, particularly when it comes to the excessive residue in fruits and herbal remedies. One of the key elements influencing the export of TCM resources is exogenous pollution, which includes PGR. Therefore, it is of great importance to investigate the potential toxicity of PGR in order to ensure human health and provide fundamental basis for the guiding of standardized and scientific application of PGR.

Plant growth regulators fall into five general categories: auxin, cytokinin, gibberellins, ethylene, and abscisic acid. Currently, the most active cytokinin synthesized is forchlorfenuron, also known as 1-(2-chloro-4-pyridyl)-3-phenylurea, which accelerates cell mitosis, promotes cell development and differentiation and prevents fruit falling. Forchlorfenuron is typically utilized in the cultivation of fruits such as watermelon, grapes, and kiwifruit, as well as herbal remedies such as Pueraria lobata, Schisandra seeds, Rhizoma Dioscoreae etc. ¹ Forchlorfenuron was found to produce fast and reversible cytokinesis abnormalities and septin filament distortion in budding yeast in 2004 ². This is the reason why forchlorfenuron has also been used to study the roles that septins play in several mammalian cellular processes. The results showed that forchlorfenuron interfered with cell proliferation, inhibited cell migration and invasion, and changed growth factor receptor signaling ^3–7. It is noteworthy that a recent study revealed that forchlorfenuron caused cell cycle arrest and cell death in addition to inhibiting the growth of malignant mesothelioma cells in humans and mice both in vitro and in vivo ⁸. Despite the fact that forchlorfenuron is thought to have a low level of toxicity, there have been reports of observable off-target effects ⁹. Previous studies demonstrated that forchlorfenuron caused cytotoxicity, cytoskeleton destruction, and decreased expression of corresponding proteins (Myl7, Gata4, and Mef2c) in H9c2 cardiomyocytes in vitro. It also caused cardiac morphology deformation, cardiac contractile dysfunction, and erythrocyte reduction in zebra fish ¹⁰. Bu et al. ¹¹ discovered that forchlorfenuron may have negative effects on steroid genesis and the ovaries in their 180-day repeated-dose toxicity study in Sprague-Dawley rats. According to research by Lei Sun et al. ¹², forchlorfenuron significantly reduces migration and weakens human epithelial cells' barrier qualities in a way that is independent of the septin cytoskeleton. Short-term toxicity studies indicated that the target organs of forchlorfenuron were the liver, the kidneys and the hematological parameters ¹³.

While retaining liver-specific functions including drug-metabolizing enzymes, the HepaRG cell line is equally susceptible to in vitro toxicity studies as human primary hepatocytes and HepG2 cells. To further shed light on the toxicity of forchlorfenuron, we use HepaRG cells to study its hepatotoxicity ¹⁴. Apoptosis is one of the most common forms of cell death in exogenous chemical-induced toxicity in the liver, which is modulated exquisitely by different intracellular protein kinases cascade. In this study, network pharmacology and cellular experiments are applied to explore the possible molecular mechanisms of forchlorfenuron-induced apoptosis in hepatocytes.

Effects of forchlorfenuron on HepaRG cell viabilities

TT assays were used to investigate the cytotoxic effects of forchlorfenuron on HepaRG cells. Results showed that forchlorfenuron inhibited cell viabilities in a time-concentration manner (Fig 1). As shown in Table 1, the IC50 values for HepaRG cells of forchlorfenuron at 24h，48h and 72h were 149.5μM, 109.6μM and 100.5μM separately. Therefore, we choose 200, 100 and 50μM for further investigation of the toxic mechanism of forchlorfenuron.

Table1．Concentration-effect equation of forchlorfenuron on HepaRG cells

Time	Equation	IC50(μM)	R²
24h	Y=100/(1+10^{(2.175-X)*(-1.398)})	149.5	0.8848
48h	Y=100/(1+10^{(2.040-X)*(-1.636)})	109.6	0.9334
72h	Y=100/(1+10^{(2.002-X)*(-1.690)})	100.5	0.9633

Effects of forchlorfenuron on HepaRG cell apoptosis

Hoechst 33342 staining and Annexin V-FITC/PI staining for ﬂow cytometry analysis were conducted to explore the apoptosis induced by forchlorfenuron incubation. As shown in Fig 2A, normal HepaRG cells exhibited homogeneous ﬂuorescence intensity of nuclei, while heterogeneous intensity and chromatin condensation of nuclei appeared in forchlorfenuron treated cells. Quantitative analysis by ﬂow cytometry showed that apoptosis rate increased in a concentration-related manner (Fig2B, Fig2C).

Effects of forchlorfenuron on ROS level in HepaRG cell

DCFH-DA staining was used to explore intracellular ROS level. As shown in Fig 3, normal cells exhibited low density of green fluorescence. After 24h incubation of forchlorfenuron, there were significant increase in intracellular ROS level, suggesting the induction of oxidative stress by forchlorfenuron.

Effects of forchlorfenuron on HepaRG cell mitochondrial membrane potential (Δψm)

Mitochondrial membrane potential depolarization was considered to be the early signs of activation of mitochondrial pathway activation. As shown in Fig 4, in control group, most cells exhibited red fluorescence, indicating the aggregates state of JC-1 inside mitochondria. While, along with the increase concentration of forchlorfenuron, the number of cells with green fluorescence also increased, suggesting that forchlorfenuron caused the depolarization of mitochondrial transmembrane potential.

Network pharmacology analysis

The results of network pharmacology indicated that the liver injury caused by forchlorfenuron involved a number of pathways and targets. A total of 50,487 liver damage targets and 694 forchlorfenuron targets were found through database searches. Forchlorfenuron and liver injury were shown to be associated with 428 genes in total (Figure 5A). A protein-protein interaction (PPI) network was created by importing these 428 genes into the STRING database, and then the results were imported in the Cytoscape software (version 3.9.1) (Figure 5B). Metascape website showed that the biological functions of 428 shared targets were revealed based on GO enrichment results in biological processes, cellular components, and molecular functions. The primary functions of the target genes were protein phosphorylation, MAPK cascade regulation, and protein kinase binding. The KEGG pathway results showed that the genes were significantly enriched in the MAPK signaling pathway, the PI3K-Akt signaling pathway, Ras signaling pathway and apoptosis (Figure 5C).

Molecular docking analysis

Molecular docking was used to establish the binding potential of forchlorfenuron to important targets of the MAPK and PI3K-Akt signaling cascade. According to the molecular docking experiments, forchlorfenuron might bind steadily to P38 and JNK, two important target proteins in the MAPK signaling cascade linked to apoptosis. Their binding energies were -7.06 kcal/mol and -6.03 kcal/mol, respectively (Figure 5D). The binding energy of Akt protein to forchlorfenuron was larger than -5 kcal/mol, and its binding capacity was comparatively low. In conclusion, forchlorfenuron might damage liver tissue through the MAPK signaling pathway based on the intermolecular interactions investigated by molecular docking and the predicted binding types and affinities of those interactions.

Effects of forchlorfenuron on p38 and JNK activation in HepaRG cells

P38 and JNK pathway plays important roles in stress-induced apoptosis in liver cell injuries. To investigate the time-course of p38 and JNK pathway activation upon forchlorfenuron treatment, western blot analysis was used to examine the protein expression of p-p38 and p-JNK at 1, 3, 6, 9, 24 and 48h. Results (Fig 6) showed that both p38 and JNK pathway were activated upon forchlorfenuron incubation. The phosphorylation of p38 reached peak at 24 h and decreased at 48h, while expression of p-JNK peaked at 48h.

Effects of forchlorfenuron on MKK3/p38/ATF-2 pathway in HepaRG cells

To further confirm the role p38 MAPK pathway in forchlorfenuron hepatotoxic effects, we examined both the upper stream regulator MKK3 and its downstream substrate transcription factor 2 (ATF-2). As shown in Fig 7, both p-MKK3 and p- ATF-2 expression increased in forchlorfenuro-teated HepaRG cells. Also, anti-apoptotic protein bcl-2 expression was inhibited and pro-apoptotic protein bax was induced, resulting in the decrease of bcl-2/bax ratio by forchlorfenuron treatment.

Effects of p38 inhibitor SB203580 on forchlorfenuron-induced cytotoxicity

As shown in Fig 8C, SB203580 alone inhibited HepaRG cell viabilities, and forchlorfenuron reduced cell viabilities in a concentration-dependent manner. Pretreatment with SB203580 reversed the cytotoxic effects of forchlorfenuron: cells in 100 μM and 50 μM groups pretreated with SB203580 exhibited significantly higher viabilities compared to cells treated with the same concentration of forchlorfenuron alone. Cells in 200 μM treated with and without SB203580 showed no difference in viabilities, which might be related to the seriousness of cell toxicities.

Effects of p38 inhibitor SB203580 on forchlorfenuron-induced apoptosis

Consistent with the results of cell viabilities, SB203580 alone induced HepaRG cell apoptosis. Apoptosis rate in 200 μM and 100 μM groups pretreated with SB203580 was significantly lower than in the group treated with forchlorfenuron alone, indicating that HepaRG cell apoptosis induced by forchlorfenuron was directly related to the p38 MAPK pathway (Fig 8A, B).

The present study demonstrated that forchlorfenuron induced HepaRG cell toxicity, caused cellular apoptosis due to the activation of the mitochondrial pathway. The MAPK signaling pathway might be involved in forchlorfenuron-induced hepatotoxicity, based on the results of network pharmacology and molecular docking. The cytotoxic effects of forchlorfenuron were further confirmed by cellular tests to link the MKK3/p38/ATF-2 pathway. The application of the p38 inhibitor SB203580 reduced cellular damage, indicating the participation of the p38 MAPK pathway.

Mitogen-activated protein kinase (MAPK) family proteins exert a significant role in regulating cell survival and death, including apoptosis ¹⁵. The extracellular signal-regulated kinases (ERKs) generally contribute to proliferation and growth, whereas p38 and c-jun N-terminal kinase (JNK) are involved in cell death ¹⁶. A wide range of extracellular ligands and stresses triggers the activation of p38 Mitogen-Activated protein kinase. The p38 MAPK undergoes dual phosphorylation at Tyr180 and Thr182 in the Thr–Gly–Tyr activation loop through the action of the upstream kinases MAPKK3 (MKK3) and MAPKK6 (MKK6) ¹⁷. Upon activation, p38 phosphorylates a wide variety of substrates in the cytoplasm and nucleus, including transcription factor 2 (ATF-2) ^18,19. Bcl-2 family members, including anti-apoptotic (e.g., Bcl-2, Bcl-xL) and pro-apoptotic (e.g., Bax, Bid) proteins, have been reported to be involved in the mitochondrial pathway ²⁰. Bcl-2/ bax ratios were crucial to determine whether the cell undergoes survival or apoptosis ²¹. Bcl-2, one of the most potent inhibitors of apoptosis, contains a CRE element responsive to ATF-2 in its promoter region ²². In our research, we intended to explore the role of MKK3/p38/ATF-2 pathway in the hepatotoxicity of forchlorfenuron in vitro.

As for in vitro cytotoxicity, previous research reported that the IC50 concentration of forchlorfenuron treatment for 48h in normal Chinese hamster ovary cells (CHO) was 12.12 ± 2.14µM ²³. For granulosa cells (GCs), 48h of 320 and 640 µM forchlorfenuron incubation significantly reduces the overall viabilities and 160µM and above concentration of forchlorfenuron exhibited higher cytotoxicity in H295R cells than in GCs at the same concentration ¹¹; For H9c2 cardiomyocytes, 24h of forchlorfenuron treatment at concentrations of 5µg/ml or higher significantly increased cell death ¹⁰. The present study demonstrated that forchlorfenuron induced HepaRG cell toxicity in a concentration- and time-dependent manner. The IC50 values for HepaRG cells of forchlorfenuron at 24h, 48h, and 72h were 149.5µM, 109.6µM, and 100.5µM separately, suggesting its potential hepatotoxicity in mammalian cells. Apoptosis is one of the most common forms of cell death and has been widely investigated in hepatotoxicity induced by various toxins and stimulators ²⁴. Apoptosis is characterized by cell shrinkage, DNA fragmentation, and destruction of nuclear proteins and cytoskeleton, etc. In our research, forchlorfenuron caused a significant increase in apoptosis and the disruption of mitochondrial membrane potential indicated that forchlorfenuron activated the intrinsic pathway of apoptosis. Bcl-2 family proteins are important regulators of mitochondrial permeability ²⁵. Our results showed that forchlorfenuron decreased the expression of anti-apoptotic protein bcl-2 and increased pro-apoptotic protein bax expression, thus decreasing the bcl-2/ bax ratio. Therefore, forchlorfenuron might cause hepatocyte injuries by inducing apoptosis through the mitochondrial pathway.

The interaction between oxidative stress and apoptosis has been profoundly investigated. In our research, forchlorfenuron incubation caused increased ROS levels in HepaRG cells, indicating the involvement of oxidative stress in the hepatoxicity of forchlorfenuron. Over-production of ROS directly causes damage to DNA, proteins, and lipids and induces cellular death via activation of different signaling pathways such as JNK and p38 mitogen-activated protein kinase. Our results showed that both JNK and P38 pathways were activated at an earlier stage upon forchlorfenuron incubation, 1h and 3h separately. Also, p38 activation peaked at 24h and subsided at 48h, while JNK activation sustained from 9 h and peaked at 48h, suggesting the involvement of the two pathways in different phases of cytotoxicity of forchlorfenuron. The time-course difference between JNK and P38 activation was consistent with the previous report ²⁶. The use of p38 inhibitor SB203580 partially reduced the cytotoxicity in HepaRG cells, further confirming the role p38 pathway in the hepatoxicity of forchlorfenuron. Following investigation demonstrated that forchlorfenuron incubation increased the activation of upstream MKK3 in a concentration-dependent way, and the molecular docking outcomes further validate this assertion (see Supplementary Fig. S1 online). The mitogen-activated protein kinase kinase 3 (MAP2K3, MKK3) is a member of the dual-specificity protein kinase group (MKK) that belongs to the MAPK signaling pathway, which phosphorylates specifically p38MAPK upon activation. In recent research, MKK3 has also been discovered to be an attractive therapeutic target in different types of cancer; however, the final biological outcome of MKK3 varies due to the pleiotropic nature of the p38 MAPK pathway ²⁷. MKK3 activation caused by oxidative stress stimulation has also been reported in other liver injuries ^28–30. Activated p38 MAPK phosphorylates a series of substrates such as transcription factor ATF-2, a component of the AP-1 transcription factor, which binds to specific target gene promoters and regulates target genes by histone modification ³¹. There were conflicting reports about the role of ATF-2 in cell survival and death ³². Ma et al reported that anti-apoptotic protein bcl-2 contains a CRE element responsive to ATF-2 in its promoter region and the absence of ATF-2 corresponded to the reduction of bcl-2 protein level in chondrocytes ²². However, there was also a research showed that the inhibition of Bcl-2 expression is regulated by histone modification with upregulation of H3K9 trimethylation via activating ATF-2 in in MDA-MB-231 cells ³³. Interestingly, in our research, forchlorfenuron significantly increased p-ATF-2 expression and decreased bcl-2 expression. The role of ATF-2 in forchlorfenuron-induced hepatocyte injuries might need more investigation.

In conclusion, plant growth regulator forchlorfenuron might cause in vitro hepatocyte toxicities. Forchlorfenuron enhanced intracellular ROS levels in HepaRG cells, which activated the mitochondrial pathway of apoptosis via activating the MKK3/p38 /ATF-2 pathway.

Materials

Forchlorfenuron was purchased from Sigma-Aldrich (St. Louis, MO, USA). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), was a product of Sigma- Aldrich (St. Louis, MO, USA). Annexin V-FITC/PI Apoptosis kit was obtained from Sigma-Aldrich (St. Louis, MO, USA). P38 inhibitor SB203580 and primary antibodies (p-38, p-p38, MKK3, p-MKK3, p-ATF2, ATF2, bcl-2, bax) were bought from Cell Signaling Technology (Danvers, MA, USA).

Data collection of network pharmacology

The Canonical SMILES of forchlorfenuron (CAS NO. 68157-60-8) were obtained from the PubChem website (https://pubchem.ncbi.nlm.nih.gov/). Then, the Swiss Target Prediction Database (http://www.swisstargetprediction.ch) and the PharmMapper database (http://www.lilab-ecust.cn/pharmmapper) were used to collect potential targets for forchlorfenuron. “Chemical and Drug Induced Liver Injury” was designated as keyword to search for hepatotoxicity targets on CTD websites (http://ctdbase.org/) ³⁴. The genes were standardized by using the Uniprot database (https://www.uniprot.org) and the intersecting targets were collected on the site venny2.1 (https://bioinfogp.cnb.csic.es/tools/venny/). The overlapped targets were integrated into the protein-protein interaction (PPI) network using the STRING database and the minimum required interaction score was set “≧0.9”. Then the intersecting targets were imported into Cytoscape 3.9.1 software and Metascape (https://metascape.org/gp/index.html#/main/step1) for obtaining the Gene Ontology (GO) functional annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively.

Molecular docking

The file containing forchlorfenuron mol2 was obtained from the Pubchem website. The PDB structures for the apoptotic pathway targets Akt, P38, MKK3 and JNK were obtained from the pdb database. Autodock 4.2.6 was used to create the molecular docking model, and Pymol was used to store the final result. By using the Autodock software, one could store the Binding Energy data, export the docking file, perform molecular docking, hydrogenation, and charge operations on compounds and proteins, and then move on to visualization and analysis.

Cell Culture

HepaRG cells were obtained from Guan Dao Biotechnology (Shanghai, China) and cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin and 100 µg/mL streptomycin. The media and supplements were purchased from Gibco Life Technologies (Grand Island, NY, USA). The cell cultures were kept in a humidified atmosphere containing 5% CO₂ at 37 ℃. Forchlorfenuron was dissolved in DMSO and incubated in FBS-free complete medium with a final DMSO concentration of 1%.

Cell Viability

Cell viability was determined using MTT assay. Briefly, HepaRG cells were plated on 96-well plates at a density of 8×10⁴ cells per well and then incubated at 37 ℃ for 24 h. The cells were treated with forchlorfenuron for 24 h, 48h and 72 h. MTT (5mg/mL) was added to each well and incubated for 4 h. The medium was removed, and the formazan crystals were dissolved with dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm on a microplate reader (TECAN Infinite M1000, Grödig, Austria).

Morphological assessment and quantification of apoptotic HepaRG cells

Hoechst 33342 staining was used for the morphological analyses of the apoptosis of cells. Apoptotic cells exhibit nuclear chromatin condensation and fragmentation. After treatment, the cells were incubated with 5 mg/ml Hoechst33342 for 15 min, washed twice with phosphate-buffered saline (PBS), and visualized by fluorescence microscopy (Leica, Germany).

Flow cytometric detection of apoptosis

After the cells were treated with indicated concentration of forchlorfenuron, apoptosis rate was evaluated with Annexin V-FITC/PI Apoptosis kit according to the manufacturer’s brochures. In brief, the cells were harvested, washed twice with cold PBS, incubated with the 5µl FITC-Annexin V and 1µl PI working solution (100µg/ml) for 15 min in the dark at room temperature, and then cellular fluorescence was measured by flow cytometry analysis with a FACSCalibur Flow Cytometer (BD Biosciences, USA).

Detection of intracellular ROS production

The production of intracellular ROS was analyzed using Reactive Oxygen Species detection kit according to the manufacturer’s brochures (Invitrogen, California, USA). Briefly, cells were washed with 1× wash buffer after treatment, and then ROS detection solution was added. The cells were stained at 37℃ in the dark for 30 min and visualized by fluorescence microscopy (Leica, Germany).

Determination of mitochondrial transmembrane potential (Δψm)

5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) (Invitrogen, USA) was used to determine the changes in mitochondrial trans-membrane potential. 10µl of 200µM JC-1 (2 µM final concentration) was added to the cells and incubated for 30 min in the dark and washed twice with PBS. The cells labeled with JC-1 were observed under fluorescence microscopy (Leica, Germany).

Western blot analysis

Cultured HepaRG cells were harvested, washed with PBS, and lysed with cell lysis buffer containing 1% phenylmethylsulfonylfluoride. The lysate was centrifuged at 12,000g for 15 min to remove the insoluble materials. Supernates were collected. Primary antibodies (p-38, p-p38, JNK, p-JNK, MKK3, p-MKK3, p-ATF2, ATF2, bcl-2, bax) from were used for indicated proteins.

Statistics

All data were expressed as the means ± SD of at least three independent experiments. Significant differences between the groups were determined by ANOVA. The level of significance was set at p < 0.05.

Data availability

The data generated during the current study are available from the corresponding author on reasonable request.

Author contributions statement

L.Y. and S.X.B. conceived and designed the project. S.X. and Z.Y.Q. led the in vitro studies with assistance. C.X.Y. analyzed the data. S.X. and Z.Y.Q. wrote the manuscript text and prepared the figures. All authors approved the submitted version.

Competing interests

All the authors declare no competing interests.

Ricci, A. & Bertoletti, C. Urea derivatives on the move: cytokinin-like activity and adventitious rooting enhancement depend on chemical structure. Plant Biol (Stuttg) 11, 262–272, DOI: https://doi.org/10.1111/j.1438-8677.2008.00165.x (2009).
Iwase, M., Okada, S., Oguchi, T. & Toh-e, A. Forchlorfenuron, a phenylurea cytokinin, disturbs septin organization in Saccharomyces cerevisiae. Genes Genet Syst 79, 199–206, DOI: https://doi.org/10.1266/ggs.79.199 (2004).
Hu, Q., Nelson, W. J. & Spiliotis, E. T. Forchlorfenuron alters mammalian septin assembly, organization, and dynamics. J Biol Chem 283, 29563–29571, DOI: https://doi.org/10.1074/jbc.M804962200 (2008).
Vardi-Oknin, D., Golan, M. & Mabjeesh, N. J. Forchlorfenuron disrupts SEPT9_i1 filaments and inhibits HIF-1. PLoS One 8, e73179, DOI: https://doi.org/10.1371/journal.pone.0073179 (2013).
Zhang, N. et al. The requirement of SEPT2 and SEPT7 for migration and invasion in human breast cancer via MEK/ERK activation. Oncotarget 7, 61587–61600, DOI: https://doi.org/10.18632/oncotarget.11402 (2016).
Sidhaye, V. K., Chau, E., Breysse, P. N. & King, L. S. Septin-2 mediates airway epithelial barrier function in physiologic and pathologic conditions. Am J Respir Cell Mol Biol 45, 120–126, DOI: https://doi.org/10.1165/rcmb.2010-0235OC (2011).
Marcus, E. A. et al. Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells. Biochem J 473, 1703–1718, DOI: https://doi.org/10.1042/BCJ20160203 (2016).
Blum, W. et al. The phytohormone forchlorfenuron decreases viability and proliferation of malignant mesothelioma cells in vitro and in vivo. Oncotarget 10, 6944–6956, DOI: https://doi.org/10.18632/oncotarget.2734127341 (2019).
Heasley, L. R., Garcia, G., 3rd & McMurray, M. A. Off-target effects of the septin drug forchlorfenuron on nonplant eukaryotes. Eukaryot Cell 13, 1411–1420, DOI: https://doi.org/10.1128/EC.00191-14 (2014).
Gong, G., Kam, H., Tse, Y. & Lee, S. M. Cardiotoxicity of forchlorfenuron (CPPU) in zebrafish (Danio rerio) and H9c2 cardiomyocytes. Chemosphere 235, 153–162, DOI: https://doi.org/10.1016/j.chemosphere.2019.06.027 (2019).
Bu, Q. et al. 180 Day Repeated-Dose Toxicity Study on Forchlorfenuron in Sprague-Dawley Rats and Its Effects on the Production of Steroid Hormones. J Agric Food Chem 67, 10207–10213, DOI: https://doi.org/10.1021/acs.jafc.9b03855 (2019).
Sun, L. et al. A Septin Cytoskeleton-Targeting Small Molecule, Forchlorfenuron, Inhibits Epithelial Migration via Septin-Independent Perturbation of Cellular Signaling. Cells 9, DOI: https://doi.org/10.3390/cells9010084 (2019).
Arena, M. et al. Peer review of the pesticide risk assessment of the active substance forchlorfenuron. EFSA journal. European Food Safety Authority 15, e04874, DOI: https://doi.org/10.2903/j.efsa.2017.4874 (2017).
Yokoyama, Y. et al. Comparison of Drug Metabolism and Its Related Hepatotoxic Effects in HepaRG, Cryopreserved Human Hepatocytes, and HepG2 Cell Cultures. Biol Pharm Bull 41, 722–732, DOI: https://doi.org/10.1248/bpb.b17-00913 (2018).
Cross, T. G. et al. Serine/threonine protein kinases and apoptosis. Exp Cell Res 256, 34–41, DOI: https://doi.org/10.1006/excr.2000.4836S0014-4827(00)94836-5 (2000).
Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J. & Greenberg, M. E. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326–1331, DOI: https://doi.org/10.1126/science.270.5240.1326 (1995).
Kang, Y. J., Seit-Nebi, A., Davis, R. J. & Han, J. Multiple activation mechanisms of p38alpha mitogen-activated protein kinase. J Biol Chem 281, 26225–26234, DOI: https://doi.org/10.1074/jbc.M606800200 (2006).
Rouse, J. et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78, 1027–1037, DOI: https://doi.org/10.1016/0092-8674(94)90277-1 (1994).
Raingeaud, J. et al. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270, 7420–7426, DOI: https://doi.org/10.1074/jbc.270.13.7420 (1995).
Antonsson, B. & Martinou, J. C. The Bcl-2 protein family. Exp Cell Res 256, 50–57, DOI: https://doi.org/10.1006/excr.2000.4839 S0014-4827(00)94839-0 (2000).
Oltersdorf, T. et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677–681, DOI: https://doi.org/10.1038/nature03579 (2005).
Ma, Q. et al. Activating transcription factor 2 controls Bcl-2 promoter activity in growth plate chondrocytes. J Cell Biochem 101, 477–487, DOI: https://doi.org/10.1002/jcb.21198 (2007).
Zhang, Z. et al. Identification, synthesis, and safety assessment of forchlorfenuron (1-(2-chloro-4-pyridyl)-3-phenylurea) and its metabolites in kiwifruits. J Agric Food Chem 63, 3059–3066, DOI: https://doi.org/10.1021/acs.jafc.5b01100 (2015).
Schwabe, R. F. & Luedde, T. Apoptosis and necroptosis in the liver: a matter of life and death. Nat Rev Gastroenterol Hepatol 15, 738–752, DOI: https://doi.org/10.1038/s41575-018-0065-y (2018).
Delbridge, A. R., Grabow, S., Strasser, A. & Vaux, D. L. Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies. Nat Rev Cancer 16, 99–109, DOI: https://doi.org/10.1038/nrc.2015.17 (2016).
Wang, S., Wang, M., Tian, Y., Sun, X. & Sun, G. Bavachinin Induces Oxidative Damage in HepaRG Cells through p38/JNK MAPK Pathways. Toxins (Basel) 10, DOI: https://doi.org/10.3390/toxins10040154 (2018).
Stramucci, L. et al. MKK3 sustains cell proliferation and survival through p38DELTA MAPK activation in colorectal cancer. Cell Death Dis 10, 842, DOI: https://doi.org/10.1038/s41419-019-2083-2 (2019).
Niino, T. et al. A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis. Biochem Pharmacol 155, 182–197, DOI: https://doi.org/10.1016/j.bcp.2018.06.021 (2018).
Hsieh, C. C., Rosenblatt, J. I. & Papaconstantinou, J. Age-associated changes in SAPK/JNK and p38 MAPK signaling in response to the generation of ROS by 3-nitropropionic acid. Mech Ageing Dev 124, 733–746, DOI: https://doi.org/10.1016/s0047-6374(03)00083-6 (2003).
Hsieh, C. C. & Papaconstantinou, J. The effect of aging on p38 signaling pathway activity in the mouse liver and in response to ROS generated by 3-nitropropionic acid. Mech Ageing Dev 123, 1423–1435, DOI: https://doi.org/10.1016/s0047-6374(02)00084-2 (2002).
Saleh, A., Alvarez-Venegas, R. & Avramova, Z. Dynamic and stable histone H3 methylation patterns at the Arabidopsis FLC and AP1 loci. Gene 423, 43–47, DOI: https://doi.org/10.1016/j.gene.2008.06.022 (2008).
Vlahopoulos, S. A. et al. The role of ATF-2 in oncogenesis. Bioessays 30, 314–327, DOI: https://doi.org/10.1002/bies.20734 (2008).
Hsieh, C. J. et al. Arctigenin, a dietary phytoestrogen, induces apoptosis of estrogen receptor-negative breast cancer cells through the ROS/p38 MAPK pathway and epigenetic regulation. Free Radic Biol Med 67, 159–170, DOI: https://doi.org/10.1016/j.freeradbiomed.2013.10.004 (2014).
Liao, Y., Ding, Y., Yu, L., Xiang, C. & Yang, M. Exploring the mechanism of Alisma orientale for the treatment of pregnancy induced hypertension and potential hepato-nephrotoxicity by using network pharmacology, network toxicology, molecular docking and molecular dynamics simulation. Frontiers in pharmacology 13, 1027112, DOI: https://doi.org/10.3389/fphar.2022.1027112 (2022).

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Forchlorfenuron exposure Induces hepatocyte Apoptosis via MKK3/p38/ATF-2 signaling Pathway

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Methods

Materials

Data collection of network pharmacology

Molecular docking

Cell Culture

Cell Viability

Morphological assessment and quantification of apoptotic HepaRG cells

Flow cytometric detection of apoptosis

Detection of intracellular ROS production

Determination of mitochondrial transmembrane potential (Δψm)

Western blot analysis

Statistics

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1