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ChaC glutathione specific γ-glutamylcyclotransferase 1 inhibits cell viability and increases the sensitivity of prostate cancer cells to docetaxel by inducing endoplasmic reticulum stress and ferroptosis.

ABSTRACT: The present study aimed to determine the effects and mechanism of ChaC glutathione specific γ-glutamylcyclotransferase 1 (CHAC1) on cell viability and the sensitivity of prostate cancer cells to docetaxel. Compared with non-tumor human prostate epithelial RWPE-1 cells, the mRNA and protein levels of CHAC1 significantly decreased in two prostate cancer cell lines, DU145 and 22RV1, as measured by quantitative polymerase chain reaction and western blot analysis (P<0.05). The cell viability and glutathione (GSH) levels were significantly inhibited in prostate cancer cells following overexpression of CHAC1 (P<0.01), while they were significantly increased in DU145 cells transfected with CHAC1 siRNA (P<0.05), but not in 22RV1 cells (P>0.05). The expression levels of several endoplasmic reticulum (ER) stress-related factors were then measured by western blot analysis. Following transfection with plasmid overexpressing CHAC1, ER markers, BIP and CHOP levels, were significantly upregulated (P<0.01), while GSH co-treatment decreased this upregulation. In addition, CHAC1 protein levels were significantly upregulated in cells treated with a ferroptosis activator (P<0.05). A liperflo reagent was then used to determine intracellular lipid peroxide levels. The intracellular lipid peroxides levels were significantly increased following CHAC1-overexpression (P<0.05), while GPX4 protein levels were significantly decreased (P<0.01). The cell viability was significantly inhibited (P<0.001) even with 1 nM docetaxel (DTX) and a plasmid overexpressing CHAC1, while the effect of inhibition was not significant at 1 nM of DTX alone (P>0.05). This inhibition was also eliminated following the addition of a ferroptosis inhibitor. In summary, CHAC1 may inhibit cell viability and increase the sensitivity of prostate cancer cells to DTX. The cellular mechanism may involve the induction of ER stress and ferroptosis. The results of the present study identified a potentially novel therapeutic target for prostate cancer, which may be useful in patients with castration-resistant prostate cancer.

SUBMITTER: He S

PROVIDER: S-EPMC8311285 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC.MethodsRNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy.ResultsRNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05).ConclusionsOur evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

Project description:AimsS-glutathionylation of cysteine residues, catalyzed by glutathione S-transferase Pi (GSTP), alters structure/function characteristics of certain targeted proteins. Our goal is to characterize how S-glutathionylation of proteins within the endoplasmic reticulum (ER) impact cell sensitivity to ER-stress inducing drugs.ResultsWe identify GSTP to be an ER-resident protein where it demonstrates both chaperone and catalytic functions. Redox based proteomic analyses identified a cluster of proteins cooperatively involved in the regulation of ER stress (immunoglobulin heavy chain-binding protein [BiP], protein disulfide isomerase [PDI], calnexin, calreticulin, endoplasmin, sarco/endoplasmic reticulum Ca2+-ATPase [SERCA]) that individually co-immunoprecipitated with GSTP (implying protein complex formation) and were subject to reactive oxygen species (ROS) induced S-glutathionylation. S-glutathionylation of each of these six proteins was attenuated in cells (liver, embryo fibroblasts or bone marrow dendritic) from mice lacking GSTP (Gstp1/p2-/-) compared to wild type (Gstp1/p2+/+). Moreover, Gstp1/p2-/- cells were significantly more sensitive to the cytotoxic effects of the ER-stress inducing drugs, thapsigargin (7-fold) and tunicamycin (2-fold).InnovationWithin the family of GST isozymes, GSTP has been ascribed the broadest range of catalytic and chaperone functions. Now, for the first time, we identify it as an ER resident protein that catalyzes S-glutathionylation of critical ER proteins within this organelle. Of note, this can provide a nexus for linkage of redox based signaling and pathways that regulate the unfolded protein response (UPR). This has novel importance in determining how some drugs kill cancer cells.ConclusionsContextually, these results provide mechanistic evidence that GSTP can exert redox regulation in the oxidative ER environment and indicate that, within the ER, GSTP influences the cellular consequences of the UPR through S-glutathionylation of a series of key interrelated proteins. Antioxid. Redox Signal. 26, 247-261.

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ChaC glutathione specific γ-glutamylcyclotransferase 1 inhibits cell viability and increases the sensitivity of prostate cancer cells to docetaxel by inducing endoplasmic reticulum stress and ferroptosis.

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