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ZIP4 confers resistance to zinc deficiency-induced apoptosis in pancreatic cancer.

ABSTRACT: Emerging evidence implicates the zinc importer ZIP4 as a critical factor that enhances pancreatic cancer proliferation; however, the role of ZIP4 in promoting pancreatic cancer progression by regulating apoptosis requires elucidation. To determine the effect of ZIP4 on apoptosis, we used cell lines where ZIP4 levels were upregulated or silenced in combination with Chelex 100 treatment to deplete intracellular zinc. Pancreatic cancer xenografts derived from those cells were also included. TUNEL and flow cytometry analysis were used to measure apoptosis and western blotting was used to analyze protein expression for PARP and multiple caspases. Cell cycle profiles were examined by flow cytometry. Zinc depletion by Chelex induced more apoptosis of pancreatic cancer cells in comparison to normal medium, where almost no apoptosis was observed. ZIP4 stably overexpressed MIA PaCa-2 (MIA-ZIP4) cells were more resistant to zinc depletion-induced apoptosis compared with vector control. Conversely, AsPC-1 (AsPC-shZIP4) cells with stable knockdown of ZIP4 were more sensitive to zinc deficiency than control. Resistance to apoptosis mediated by ZIP4 was accomplished by the caspase pathway. In vivo data also confirmed that ZIP4 overexpressed xenografts showed less apoptosis than controls. Cell cycle profiles indicate that silencing of ZIP4 leads to decreased cell population in S phase and G 0/G 1 arrest. These results described a previously uncharacterized role of ZIP4 in apoptosis resistance and elucidated a novel pathway through which ZIP4 regulates pancreatic cancer growth. This research provides additional evidence for ZIP4 and related signaling cascade as a molecular target for therapeutic intervention in pancreatic cancer.

SUBMITTER: Cui X

PROVIDER: S-EPMC4013168 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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ZIP4 confers resistance to zinc deficiency-induced apoptosis in pancreatic cancer.

Cui Xiaobo X Zhang Yuqing Y Yang Jingxuan J Sun Xiaotian X Hagan John P JP Guha Sushovan S Li Min M

Cell cycle (Georgetown, Tex.) 20140211 7

Emerging evidence implicates the zinc importer ZIP4 as a critical factor that enhances pancreatic cancer proliferation; however, the role of ZIP4 in promoting pancreatic cancer progression by regulating apoptosis requires elucidation. To determine the effect of ZIP4 on apoptosis, we used cell lines where ZIP4 levels were upregulated or silenced in combination with Chelex 100 treatment to deplete intracellular zinc. Pancreatic cancer xenografts derived from those cells were also included. TUNEL a ...[more]

PMID: 24553114

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Project description:Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity.In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha.PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.

Project description:BackgroundPrevious studies showed that mesothelin (MSLN) plays important roles in survival of pancreatic cancer (PC) cells under anchorage dependent/independent conditions as well as resistance to chemotherapy. The recent success of intratumorally-injected adeno-encoded, chemo/radiation-inducible-promoter driven hTNF-?, (TNFerade) + gemcitabine in pre-clinical models of PC have renewed interest in use of TNF-? as a therapeutic component. To help find additional factors which might affect the therapy, we examined the resistance of MSLN-overexpressing pancreatic cancer cell lines to TNF-?-induced growth inhibition/apoptosis.MethodsStable MSLN overexpressing MIA PaCa-2 cells (MIA-MSLN), stable MSLN-silenced AsPC-1 cells (AsPC-shMSLN) and other pancreatic cells (MIA-PaCa2, Panc 28, Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48) were used. NF-?B activation was examined by western blots and luciferase reporter assay. TNF-? induced growth inhibition/apoptosis was measured by MTT, TUNEL assay and caspase activation. IL-6 was measured using luminex based assay.ResultsCompared to low endogenous MSLN-expressing MIA PaCa-2 and Panc 28 cells, high endogenous MSLN-expressing Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48 cells were resistant to TNF-? induced growth inhibition. Stable MSLN overexpressing MIA-PaCa2 cells (MIA-MSLN) were resistant to TNF-?-induced apoptosis while stable MSLN-silenced AsPC1 cells (AsPC-shMSLN) were sensitive. Interestingly, TNF-?-treated MIA-MSLN cells showed increased cell cycle progression and cyclin A induction, both of which were reversed by caspase inhibition. We further found that MIA-MSLN cells showed increased expression of anti-apoptotic Bcl-XL and Mcl-1; deactivated (p-Ser75) BAD, and activated (p-Ser70) Bcl-2. Constitutively activated NF-?B and Akt were evident in MIA-MSLN cells that could be suppressed by MSLN siRNA with a resultant increase in sensitivity of TNF-? induced apoptosis. Blocking NF-?B using IKK inhibitor wedelolactone also increased sensitivity to TNF-?-mediated cytotoxicity with concomitant decrease in Mcl-1. Blocking Akt using PI3K inhibitor also had a likewise effect presumably affecting cell cycle. MIA-MSLN cells produced increased IL-6 and were increased furthermore by TNF-? treatment. SiRNA-silencing of IL-6 increased TNF-? sensitivity of MIA-MSLN cells.ConclusionsOur study delineates a MSLN-Akt-NF-?B-IL-6-Mcl-1 survival axis that may be operative in PC cells, and might help cancer cells' survival in the highly inflammatory milieu evident in PC. Further, for the success of TNFerade + gemcitabine to be successful, we feel the simultaneous inhibition of components of this axis is also essential.

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ZIP4 confers resistance to zinc deficiency-induced apoptosis in pancreatic cancer.

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ZIP4 confers resistance to zinc deficiency-induced apoptosis in pancreatic cancer.

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