Increased stability of P21(WAF1/CIP1) mRNA is required for ROS/ERK-dependent pancreatic adenocarcinoma cell growth inhibition by pyrrolidine dithiocarbamate

We present evidence that pyrrolidine dithiocarbamate (PDTC) inhibits growth of p53-negative pancreatic adenocarcinoma cell lines via cell cycle arrest in the S-phase, while it has no effect on primary fibroblast proliferation. Growth inhibition of cancer cells is dependent on ROS and ERK1/2 induction as indicated by a significantly reduced PDTC-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine (NAC) or the MEK/ERK1/2 inhibitor (PD98059). Moreover, ERK1/2 induction is dependent on ROS production as demonstrated by a complete removal of PDTC-mediated ERK1/2 phosphorylation by NAC. p21(WAF1/CIP1) activation has a central role in growth inhibition by PDTC, as revealed by P21(WAF1/CIP1) silencing experiments with antisense oligonucleotide, and occurs via increased mRNA stability largely mediated by ROS/ERK induction. Conversely, PDTC does not affect P21(WAF1/CIP1) gene expression in primary fibroblasts, although it is able to activate p53 and the p53-regulated antioxidant SESN2. These results suggest that the resistance of fibroblasts to the cytotoxic action of PDTC may be related to the up-regulation of p53-dependent antioxidant genes. Finally, in vivo studies on PaCa44 cells subcutaneously xenografted in nude mice show that treatment with 100 or 200 mg/kg PDTC reduces of 30% or 60% the tumour volume, respectively, and does not cause any apparent form of toxicity.     

Gemcitabine response in pancreatic adenocarcinoma cells is synergistically enhanced by dithiocarbamate derivatives

Pancreatic adenocarcinoma is a common malignancy that remains refractory to all available therapies, including the gold standard drug gemcitabine (GEM). We investigated the effect of the combination of GEM and each of the ionophore compounds pyrrolidine dithiocarbamate (PDTC) and disulfiram [DSF; 1-(diethylthiocarbamoyldisulfanyl)-N,N-diethylmethanethioamide] on p53^?/? pancreatic adenocarcinoma cell growth. PDTC or DSF synergistically inhibited cell proliferation when used in combination with GEM by inducing apoptotic cell death. This effect was associated with an increased mitochondrial O₂^?? production and was further enhanced by zinc ions. Basal levels of mitochondrial O₂^?? or manganese superoxide dismutase (MnSOD) strictly correlated with the IC₅₀ for GEM or the percentage of synergism. Thus, the most relevant values of the antiproliferative synergism were obtained in GEM-resistant pancreatic adenocarcinoma cell lines. Interestingly, the GEM-sensitive T3M4 cells transfected with MnSOD expression vector showed mitochondrial O₂^?? and IC₅₀ for GEM similar to those of resistant cell lines. In vivo experiments performed on nude mice xenotransplanted with the GEM-resistant PaCa44 cell line showed that only the combined treatment with GEM and DSF/Zn completely inhibited the growth of the tumoral masses. These results and the consideration that DSF is already used in clinics strongly support the GEM and DSF/Zn combination as a new approach to overcoming pancreatic cancer resistance to standard chemotherapy.     

Zinc depletion efficiently inhibits pancreatic cancer cell growth by increasing the ratio of antiproliferative/proliferative genes

We investigated the ability of the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to reduce pancreatic cancer cell viability. TPEN was much more efficient to inhibit pancreatic adenocarcinoma cell growth than a panel of anti-cancer drugs, including 5-fluorouracil, irinotecan, cisplatin, edelfosine, trichostatin A, mitomycin C, and gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Moreover, TPEN showed a dose- and time-dependent anti-proliferative effect significantly higher on pancreatic cancer cells than on normal primary fibroblasts. This effect may be explained by a significantly higher zinc depletion by TPEN in pancreatic cancer cells as compared to fibroblasts. Cell viability reduction by TPEN was associated to both G1-phase cell cycle arrest and apoptosis, and to the increased ratio of the expression level of cyclin-Cdk inhibitor versus cyclin genes and apoptotic versus anti-apoptotic genes. Finally, we show that apoptotic cell death induced by TPEN involved mitochondrial injury and caspase 3 and caspase 8 activation. In this study, we suggest that zinc depletion may be an efficient strategy in the treatment of pancreatic cancer because of its reduced antiproliferative effect on normal cells.     

PDTC, metal chelating compound, induces G1 phase cell cycle arrest in vascular smooth muscle cells through inducing p21Cip1 expression: involvement of p38 mitogen activated protein kinase

Pyrrolidine dithiocarbamate (PDTC), a metal chelating compound, is known to induce cell death in vascular smooth muscle cells (VSMC). However, the molecular mechanism for PDTC-induced VSMC death is not well understood. Addition of PDTC reduced cell growth and DNA synthesis on VSMC in low density conditions. However, in serum depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. Several metal chelators prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper, iron, and zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper, iron, and zinc in serum may mediate the cytotoxic effect of PDTC. At low VSMC density in 10% FBS, treatment of PDTC, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by PDTC was not observed. Finally, we determined PDTC-mediated signaling pathway involved in VSMC death. Among relevant pathways, PDTC induced marked activation of p38MAPK and JNK. Expression of dominant negative p38MAPK and SB203580, a p38MAPK specific inhibitor, blocked PDTC-dependent p38MAPK, growth inhibition, and p21 expression. These data demonstrate that the p38MAPK pathway participates in p21 induction, which consequently leads to decrease of cyclin D1/cdk4 and cyclin E/cdk2 complexes and PDTC-dependent VSMC growth inhibition. In conclusion, an understanding of the molecular mechanisms of PDTC in VSMC provides a theoretical basis for clinical approaches using antioxidant therapies in atherosclerosis.     

Synergistic effect of trichostatin A and 5‐aza‐2′‐deoxycytidine on growth inhibition of pancreatic endocrine tumour cell lines: A proteomic study

Our research group recently reported that pancreatic endocrine cancer cell lines are sensitive to the HDAC inhibitor trichostatin A (TSA). In the present paper, we show that the combined treatment of pancreatic endocrine tumour cell lines with TSA and the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine (DAC) determines a strong synergistic inhibition of proliferation mainly due to apoptotic cell death. Proteomic analysis demonstrates that the modulation of specific proteins correlates with the antiproliferative effect of the drugs. A schematic network clarifies the most important targets or pathways involved in pancreatic endocrine cancer growth inhibition by single or combined drug treatments, which include proteasome, mitochondrial apoptotic pathway and caspase related proteins, p53 and Ras related proteins. A comparison between the patterns of proteins regulated by TSA or DAC in endocrine and ductal pancreatic cancer cell lines is also presented.     

Zinc and p53 disrupt mitochondrial binding of HK2 by phosphorylating VDAC1

Many cell death regulators physically or functionally interact with metabolic enzymes. These interactions provide insights into mechanisms of anticancer treatments from the perspective of tumor cell metabolism and apoptosis. Recent studies have shown that zinc and p53 not only induce tumor cell apoptosis, but also regulate tumor cell metabolism. However, the underlying mechanism is complex and remains unclear, making further research imperative to provide clues for future cancer treatments. In this study, we found that hexokinase 2 (HK2), which has dual metabolic and apoptotic functions, is downstream of zinc and p53 in both prostate cancer patient tissue and prostate cancer cell lines. Notably, the mitochondrial location of HK2 is crucial for its function. We demonstrate that zinc and p53 disrupt mitochondrial binding of HK2 in prostate cancer cells by phosphorylating VDAC1, which is mediated by protein kinase B (Akt) inhibition and glycogen synthase kinase 3β (GSK3β) activation. In addition, we found that zinc combined with p53 significantly inhibited tumor growth in a prostate cancer cell xenograft model. Therefore, interference of the mitochondrial localization of HK2 by zinc and p53 may provide a new treatment approach for cancer.     

Reactive oxygen species modulate Zn(2+)-induced apoptosis in cancer cells

Some recent evidence has suggested a protective role of zinc against cancer. The mechanism by which zinc exerts this action has not been defined and, in particular, it has not been clarified whether zinc may directly act on cancer cells and the molecular mechanisms involved in this effect. In this study, we examined the in vitro effect of zinc on the apoptosis of mouse TS/A mammary adenocarcinoma cells, studying the zinc-dependent modulation of the intracellular levels of reactive oxygen species (ROS) and of p53 and Fas/Fas ligand pathways. We showed that zinc concentrations ranging from 33.7 to 75 muM Zn(2+) induced apoptosis in mammary cancer cells. The apoptosis was associated with an increased production of intracellular ROS, and of p53 and Fas/Fas ligand mRNA and protein. Zn(2+) induced a faint metallothionein response in TS/A cells in comparison with mouse lymphocytes. The treatment of tumor cells with the antioxidant N-acetylcysteine was able to prevent Zn(2+)-induced apoptosis, as well as the increase of p53 and Fas ligand protein induced by zinc. The data demonstrate that zinc exerts a direct action on mammary cancer cells inducing ROS-mediated apoptosis and that the effect may be mediated by the ROS-dependent induction of p53 and Fas/Fas ligand.     

The ion channel TRPM7 regulates zinc-depletion-induced MDMX degradation

Zinc deficiency has been linked to human diseases, including cancer. MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or overexpressed in various cancers and implicated in the cancer initiation and progression. We report here that zinc depletion by the ion chelator TPEN or Chelex resin results in MDMX protein degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc led to recovery of cellular levels of MDMX. Further, TPEN treatment inhibits growth of the MCF-7 breast cancer cell line, which is partially rescued by overexpression of MDMX. Moreover, in a mass-spectrometry-based proteomics analysis, we identified TRPM7, a zinc-permeable ion channel, as a novel MDMX-interacting protein. TRPM7 stabilizes and induces the appearance of faster migrating species of MDMX on SDS-PAGE. Depletion of TRPM7 attenuates, while TRPM7 overexpression facilitates, the recovery of MDMX levels upon adding back zinc to TPEN-treated cells. Importantly, we found that TRPM7 inhibition, like TPEN treatment, decreases breast cancer cell MCF-7 proliferation and migration. The inhibitory effect on cell migration upon TRPM7 inhibition is also partially rescued by overexpression of MDMX. Together, our data indicate that TRPM7 regulates cellular levels of MDMX in part by modulating the intracellular Zn²⁺ concentration to promote tumorigenesis.     

5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation

In addition to its demethylating function, 5-aza-2'-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.     

Zinc chelator TPEN induces pancreatic cancer cell death through causing oxidative stress and inhibiting cell autophagy

The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems including immunodeficiency and sensory dysfunctions. Previous studies had shown that Zn availability was also important for tumor growth and progression. The aim of the present study was to investigate the potential mechanisms of N,N,N,N-Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (a membrane permeable zinc chelator) induced pancreatic cancer cell death. The text of inductively coupled plasma-mass spectrometry (ICP-MS) showed in human pancreatic cancer samples that the zinc content in cancer was higher than that in adjacent tissues. The pancreatic cancer cell lines Panc-1, 8988T, BxPc-3, and L3.6 were used in this study. Our results indicated that TPEN markedly induced cell death, via increasing reactive oxygen species (ROS) and restraining autophagy. Our data also indicated that TPEN-stimulated mitochondrial metabolism produced much ROS. Meanwhile, TPEN reduced the levels of glutathione (GSH) and triggered ROS outbreak, which were the main causes of cell death. In addition, cell autophagy was significantly depressed in Panc-1 cells treated by TPEN, which was due to the ability of disrupting lysosomal by TPEN. Thus, we thought zinc depletion by TPEN was a potential therapeutic strategy for pancreatic cancer.     

Pyrrolidine dithiocarbamate and zinc inhibit proteasome-dependent proteolysis

Proteasomes play important roles in a variety of cellular processes such as cell cycle progression, signal transduction and immune responses. Proteasome activity is important in maintaining rapid turnover of short-lived proteins, as well as preventing accumulation of misfolded or damaged proteins. Alteration in ubiquitin-proteasome function may be detrimental to its crucial role in maintaining cellular homeostasis. Here, we have found that treatment of pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, resulted in the accumulation of several proteasome substrates including p53 and p21 in HeLa cells. The PDTC effect was due to an extended half-life of these proteins through the mobilization of zinc. PDTC and/or zinc also increased fluorescence intensity of Ub(G76V)-GFP fusion protein that is degraded rapidly by the ubiquitin-proteasome system. Treatment of cells with zinc induced formation of ubiquitinated inclusions in the centrosome, a histological marker of proteasome inhibition. Western blotting showed zinc-induced increase in laddering bands of polyubiquitin-conjugated proteins. In vitro study, zinc inhibited the ubiquitin-independent proteasomal degradations of p21 and alpha-synuclein. These results suggest that zinc may modulate cell functions through its action on the turnover of proteins that are susceptible to proteasome-dependent proteolysis.     

Gemcitabine/cannabinoid combination triggers autophagy in pancreatic cancer cells through a ROS-mediated mechanism

Gemcitabine (GEM, 2′,2′-difluorodeoxycytidine) is currently used in advanced pancreatic adenocarcinoma, with a response rate of < 20%. The purpose of our work was to improve GEM activity by addition of cannabinoids. Here, we show that GEM induces both cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) receptors by an NF-κB-dependent mechanism and that its association with cannabinoids synergistically inhibits pancreatic adenocarcinoma cell growth and increases reactive oxygen species (ROS) induced by single treatments. The antiproliferative synergism is prevented by the radical scavenger N-acetyl--cysteine and by the specific NF-κB inhibitor BAY 11-7085, demonstrating that the induction of ROS by GEM/cannabinoids and of NF-κB by GEM is required for this effect. In addition, we report that neither apoptotic nor cytostatic mechanisms are responsible for the synergistic cell growth inhibition, which is strictly associated with the enhancement of endoplasmic reticulum stress and autophagic cell death. Noteworthy, the antiproliferative synergism is stronger in GEM-resistant pancreatic cancer cell lines compared with GEM-sensitive pancreatic cancer cell lines. The combined treatment strongly inhibits growth of human pancreatic tumor cells xenografted in nude mice without apparent toxic effects. These findings support a key role of the ROS-dependent activation of an autophagic program in the synergistic growth inhibition induced by GEM/cannabinoid combination in human pancreatic cancer cells.     

Pyrrolidine dithiocarbamate induces bovine cerebral endothelial cell death by increasing the intracellular zinc level

The antioxidant and metal-chelating effects of pyrrolidine dithiocarbamate (PDTC) have been extensively studied. PDTC prevents cell death induced by various insults. However, PDTC itself may cause cell death in selected experimental paradigms. PDTC induced bovine cerebral endothelial cell death. However, in serum-depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. The metal chelators bathocuproine disulfonic acid, o-phenanthroline, bathophenanthroline disulfonic acid, and N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN) prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper or zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper or zinc in serum may mediate the cytotoxic effect of PDTC. The potency of zinc for PDTC-induced endothelial cell death was greater than that of copper. Zn-EDTA did not block PDTC-induced cell death, whereas Ca-EDTA and Cu-EDTA were able to prevent this PDTC effect. PDTC increased the intracellular fluorescence of the zinc probe dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide, which was quenched by TPEN or various EDTA preparations but not by Zn-EDTA. Results suggest that an increase in intracellular zinc concentration is required in PDTC-induced cerebral endothelial cell death.