Ferritinophagy-mediated apoptosis and paraptosis induction involved MAPK and PI3K/AKT pathway in mechanism of an iron chelator.

Melanoma cells were more resistant to ferroptosis with still poor therapy outcomes. Sensitizing melanoma cell to the ferroptosis inducer was a crucial strategy for treatment of melanoma. In the present study, 2,2'-di-pyridylketone hydrazone dithiocarbamate s-butyric acid (DpdtbA) displayed superior inhibitory activity than ferroptosis inducer Erastin in melanoma cells, which prompt us to explore the underlying mechanism. The analyses from flow cytometry and Western blot showed that the growth inhibition of DpdtbA against SK-MEL-28 and A375 cells involved apoptosis induction and G1 phase arrest. Surprisingly, the cytoplasmic vacuoles were found upon the treatment; transmission electron microscopy and endoplasmic reticulum (ER) staining revealed that the cytoplasmic vacuoles were in ER; while down-regulation of alix and requirement of protein synthesis suggested there was an occurrence of paraptosis. However, both NAC and 3-MA could significantly attenuate the cytoplasmic vacuolization and growth inhibition, hinting that both ROS and autophagy involved the paraptosis induction. The additional evidence revealed that there was an occurrence of continuous ferritinophagy, which was responsible for the ROS production. Downregulation of NCOA4 clearly attenuated the apoptosis and paraptosis induction. In addition, activation of MAPK involved regulation of paraptosis, but only ERK and JNK had role in the formation of cytoplasmic vacuoles and growth inhibition. Furthermore, a ROS dependent regulation of PI3K/AKT pathway was also involved. Taken together, our result firstly demonstrated that a continuous ferritinophagy contributed to the apoptosis and paraptosis induction, highlighting that the lysosomal labile iron pool had a crucial role in control of melanoma cell fate.

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.

DpdtbA-Induced Growth Inhibition in Human Esophageal Cancer Cells Involved Inactivation of the p53/EGFR/AKT Pathway.

Esophageal cancer (ESC) is one of the most deadly diseases for human. p53 in most cancers, including ESC cell, is mutated, and the mutated p53 losses its original function and acquires "gain of function" that allows for promoting the hallmarks of cancer, such as antiapoptosis, metastasis, invasion, angiogenesis, and resistance to chemotherapy. Targeting p53 through either introducing wild-type or degrading mutated p53 is an important strategy in cancer therapy. Di-2,2'-pyridine ketone dithiocarbamate s-butyric acid (DpdtbA) has significant growth inhibition against gastric cancer lines in previous study. Similar action in ESC cell lines but a novel molecular mechanism was observed in the present study. The results showed that DpdtbA exhibited an excellent antiproliferative effect for ESC cell lines (IC50 ≤ 4.5 ± 0.4 µM for Kyse 450, 3.2 ± 0.6 µM for Kyse 510 cell, and 10.0 ± 0.6 µM for Kyse 150) and led to cell cycle arrest at the S phase which correlated to CDK2 downregulation. The mechanistic study suggested that growth inhibition was related to ROS-mediated apoptosis, and ROS production was due to SOD inhibition initiated by DpdtbA rather than occurrence of ferritinophagy. In addition, DpdtbA also induced a downregulation of EGFR, p53, and AKT, which hinted that mutant p53 still played a role in the regulation of its downstream targets. Further study revealed that the downregulation of p53 was through stub1- (chip-) mediated autophagic degradation rather than MDM2-mediated ubiquitination. Taken together, the DpdtbA-induced growth inhibition in a mechanism was through inactivating the p53/EGFR/AKT signal pathway.

Di-2-pyridylhydrazone Dithiocarbamate Butyric Acid Ester Exerted Its Proliferative Inhibition against Gastric Cell via ROS-Mediated Apoptosis and Autophagy.

Diversified biological activities of dithiocarbamates have attracted widespread attention; improving their feature or exploring their potent action of mechanism is a hot topic in medicinal research. Herein, we presented a study on synthesis and investigation of a novel dithiocarbamate, DpdtbA (di-2-pyridylhydrazone dithiocarbamate butyric acid ester), on antitumor activity. The growth inhibition assay revealed that DpdtbA had important antitumor activity for gastric cancer (GC) cell lines (IC50 = 4.2 ± 0.52 µM for SGC-7901, 3.80 ± 0.40 µM for MGC-803). The next study indicated that growth inhibition is involved in ROS generation in mechanism; accordingly, the changes in mitochondrial membrane permeability, apoptotic genes, cytochrome c, bax, and bcl-2 were observed, implying that the growth inhibition of DpdtbA is involved in ROS-mediated apoptosis. On the other hand, the upregulated p53 upon DpdtbA treatment implied that p53 could also mediate the apoptosis. Yet the excess generation of ROS induced by DpdtbA led to cathepsin D translocation and increase of autophagic vacuoles and LC3-II, demonstrating that autophagy was also a contributor to growth inhibition. Further investigation showed that DpdtbA could induce cell cycle arrest at the G1 phase. This clearly indicated the growth inhibition of DpdtbA was via triggering ROS formation and evoking p53 response, consequently leading to alteration in gene expressions that are related to cell survival.

Knockdown of MDR1 increases the sensitivity to adriamycin in drug resistant gastric cancer cells.

Gastric cancer is one of the most frequently occurring malignancies in the world. Development of multiple drug resistance (MDR) to chemotherapy is known as the major cause of treatment failure for gastric cancer. Multiple drug resistance 1/P-glycoprotein (MDR1/p-gp) contributes to drug resistance via ATP-dependent drug efflux pumps and is overexpressed in many solid tumors including gastric cancer. To investigate the role of MDR1 knockdown on drug resistance reversal, we knocked down MDR1 expression using shRNA in drug resistant gastric cancer cells and examined the consequences with regard to adriamycin (ADR) accumulation and drug- sensitivity. Two shRNAs efficiently inhibited mRNA and protein expression of MDR1 in SGC7901-MDR1 cells. MDR1 knockdown obviously decreased the ADR accumulation in cells and increased the sensitivity to ADR treatment. Together, our results revealed a crucial role of MDR1 in drug resistance and confirmed that MDR1 knockdown could reverse this phenotype in gastric cancer cells.

[Screening effective sequences of small interfering RNAs targeting MDR1 gene in human gastric cancer SGC7901/VCR cells].

OBJECTIVE: To screen effective sequences of small interfering RNA targeting MDR1 gene in human gastric cancer SGC7901/VCR cells. METHODS: Four siRNAs (MDR1si326, MDR1si1513, MDR1si2631 and MDR1si3071) targeting MDR1 gene were designed and synthesized by in vitro transcription. The siRNA duplexes were used to transfect into the human gastric cancer SGC7901/VCR cells. The expression level of MDR1 mRNA and P-gp were detected by RT-PCR and Western blotting, respectively. The accumulation of intracellular adriamycin (ADR) was examined by flow cytometry and the cell sensitivity to ADR was demonstrated by MTT. RESULTS: The SGC7901/VCR cells treated with 4 siRNAs led to reversal effect on multidrug resistance to different extents. Among the SGC7901/VCR cells treated by siRNAs for 48 h, the expression level of MDR1 mRNA in cells of MDR1si326 or MDR1si2631 group (0.42 +/- 0.07 or 0.49 +/- 0.02) was more decreased than that in cells of MDR1si1513 or MDR1si3071 group (P < 0.05). The accumulation of ADR in cells of MDR1si326 group was the most; in cells of MDR1si2631 group, more; in cells of MDR1si3071 group, lower and in cells of MDR1si1513 group, the lowest (P < 0.05). The relative reversal efficiency of cells of MDR1si2631 group to ADR was the highest and in cells of MDR1si326 group, higher (P < 0.05). There was no significant difference in the relative reversal efficiency between the cells of MDR1si1513 and MDR1si3071 groups (P > 0.05). The expression level of P-gp in cells of MDR1si326 group was the lowest among the SGC7901/VCR cells treated by siRNAs for 72 h. CONCLUSION: The MDR1si326 with most, MDR1si2631 with more, MDR1si3071 with less and MDR1si1513 with least reversal effects on MDR1 gene mediated multidrug resistance were found in the human gastric cancer SGC7901/VCR cells.