Ferritinophagy-Mediated Ferroptosis and Activation of Keap1/Nrf2/HO-1 Pathway Were Conducive to EMT Inhibition of Gastric Cancer Cells in Action of 2,2'-Di-pyridineketone Hydrazone Dithiocarbamate Butyric Acid Ester.

In metastasis of cancer cells, the epithelial-mesenchymal transition (EMT) is prerequired. Ferroptosis is an iron-mediated cellular death process, but whether it involves EMT regulation remains elusive. In addition, how stress responders (Nrf2) respond to the redox alteration and cross-talking between them needs to be determined. Our data revealed that DpdtbA (2,2'-di-pyridineketone hydrazone dithiocarbamate butyric acid ester) resisted TGF-ß1-induced EMT in gastric cancer lines (SGC-7901 and MGC-823) through ferritinophagy-mediated ROS production. Furthermore, the depletion of Gpx4 and xCT as well as enhanced lipid peroxidation indicated that DpdtbA acted as Erastin did in ferroptosis induction, which thus provided chance to explore the causal relationship between ferroptosis and EMT. Our data illustrated that ferritinophagy-mediated ferroptosis promoted the EMT inhibition. In addition, activated Nrf2 involved the regulation on both ferroptosis and EMT in response to the alteration in the cellular redox environment. In brief, ferritinophagy-mediated ferroptosis and activation of the Keap1/Nrf2/HO-1 pathway were conducive to the EMT inhibition.

Ferritinophagy-Mediated ROS Production Contributed to Proliferation Inhibition, Apoptosis, and Ferroptosis Induction in Action of Mechanism of 2-Pyridylhydrazone Dithiocarbamate Acetate.

Reactive oxygen species (ROS) production is involved in the mechanism of action of a number of drugs, but the biological effects of ROS remain to be clarified. Furthermore, ferroptosis involves iron-dependent ROS production that may be derived from ferritinophagy; however, the association between ferroptosis and ferritinophagy has not been fully established. The present study demonstrated that dithiocarbamate derivatives (iron chelators) exhibited antineoplastic properties involving ferritinophagy induction, but whether the underlying mechanisms involved ferroptosis was unknown. To gain insight into the underlying mechanism, a dithiocarbamate derivative, 2-pyridylhydrazone dithiocarbamate s-acetic acid (PdtaA), was prepared. An MTT assay demonstrated that PdtaA inhibited proliferation involving ROS production (IC50 = 23.0 ± 1.5 µM for HepG2 cells). A preliminary mechanistic study revealed that PdtaA induced both apoptosis and cell cycle arrest. Notably, PdtaA also induced ferroptosis via downregulation of GPx4 and xCT, which was first reported for a dithiocarbamate derivative. Moreover, these cellular events were associated with ROS production. To explore the origin of ROS, expression of the ferritinophagy-related genes, ferritin, and nuclear receptor coactivator (NCOA4) were measured. Immunofluorescence and western blotting analysis indicated that PdtaA-induced ferritinophagy may contribute to ROS production. To investigate the role of ferritinophagy, autophagy inhibitor 3-methyladenin or genetic knockdown of NCOA4 was employed to inhibit ferritinophagy, which significantly neutralized the action of PdtaA in both apoptosis and ferroptosis. Taken together, PdtaA-induced cell cycle arrest, apoptosis, and ferroptosis were associated with ferritinophagy.

The DpdtbA induced EMT inhibition in gastric cancer cell lines was through ferritinophagy-mediated activation of p53 and PHD2/hif-1α pathway.

Previous studies have shown that epithelial-mesenchymal transition (EMT) involves reactive oxygen species (ROS) production, but how ferritinophagy-mediated ROS production affects EMT status remains obscure. 2,2'-di-pyridylketone hydrazone dithiocarbamate s-butyric acid (DpdtbA), an iron chelator, exhibited interesting antitumor activities against gastric and esophageal cancer cells. As an extension of our previous research, in this paper we presented the effect of DpdtbA on EMT regulation of gastric cancer lines (SGC-7901 and MGC-803) in both normoxic and hypoxic conditions. The data from immunofluorescent and Western blotting analysis revealed that DpdtbA treatment resulted in EMT inhibition along with downregulation of hypoxia-inducible factor (hif-1α), hinting that prolyl hydroxylase 2 (PHD2) was involved. Knockdown of PHD2 significantly attenuated the action of DpdtbA on EMT regulation, supporting that PHD2 involved the EMT modulation. In addition, the inhibition of EMT involved ROS production that stemmed from DpdtbA induced ferritinophagy; while the accumulation of ferrous iron due to ferritinophagy contributed to PHD2 activation and hif-1α degradation. The correlation analysis revealed that ferritinophagic flux was a dominant driving force in determination of the EMT status. Futhermore, the ferritinophagy-mediated ROS production triggered p53 activation. Taken together, All data supported that DpdtbA induced EMT inhibition was through activation of p53 and PHD2/hif-1α pathway.

The vicious cycle between ferritinophagy and ROS production triggered EMT inhibition of gastric cancer cells was through p53/AKT/mTor pathway.

Cancer metastasis and resistance for chemotherapeutic agent correlate with epithelial-mesenchymal transition (EMT), while ROS production also involves in the EMT process, However, how autophagy mediated ROS production affects EMT remains unclear. Previous study showed that DpdtC (2,2'-di-pyridylketone hydrazone dithiocarbamate) could induce ferritinophagy in HepG2 cell. To insight into more details that how ferritinophagy affects cellular feature, the SGC-7901and BGC-823 gastric cancer cell lines were used. Interestingly DpdtC treatment resulted in EMT inhibition and was ROS dependent. Similar situation occurred in TGF-ß1 induced EMT model, supporting that DpdtC was able to inhibit EMT. Next the ability of DpdtC in ferritinophagy induction was further evaluated. As expected, DpdtC induced ferritinophagy in the absence and presence of TGF-ß1. The correlation analysis revealed that an enhanced ferritinophagic flux contributed to the EMT inhibition. In addition, ferritinophagy triggers Fenton reaction, resulting in ROS production which give rise of p53 response, thus the role of p53 was further investigated. DpdtC treatment resulted in upregulation of p53, but, the addition of p53 inhibitor, PFT-α could significantly neutralize the action of DpdtC on ferritinophagy induction and EMT inhibition. Furthermore, autophagy inhibitors or NAC could counteract the action of DpdtC, indicating that ferrtinophagy-mediated ROS played an important role in the cellular events. In addition to upregulation of p53, its down-stream targets, AKT/mTor were also downregulated, supporting that DpdtC induced EMT inhibition was achieved through ferritinophagy-ROS vicious cycle mediated p53/AKT/mTor pathway. And the activation of ferritinophagic flux was the dominant driving force in action of DpdtC in gastric cancer cells.