Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-κB in immortalized and malignant oral keratinocytes

Background  

Interleukin-8 (IL-8) is a cytokine that plays an important role in tumor progression in a variety of cancer types; however, its regulation is not well understood in oral cancer cells. In the present study, we examined the expression and mechanism of IL-8 in which it is involved by treating immortalized (IHOK) and malignant human oral keratinocytes (HN12) cells with deferoxamine (DFO).     

BRAF mutations are associated with increased iron regulatory protein‐2 expression in colorectal tumorigenesis

A role for iron in carcinogenesis is supported by evidence that iron metabolism proteins are modulated in cancer progression. To date, however, the expression of iron regulatory protein‐2 (IRP2), which is known to regulate several iron metabolism proteins, has not been assessed in colorectal cancer. Expression of IRP2 was assessed by quantitative RT‐PCR and immunohistochemistry in human colorectal cancer tissue. By interrogating The Cancer Genome Atlas (TCGA) database, expression of IRP2 and transferrin receptor‐1 (TfR1) was assessed relative to common mutations that are known to occur in cancer. The impact of suppressing IRP2 on cellular iron metabolism was also determined by using siRNA and by using the MEK inhibitor trametinib. IRP2 was overexpressed in colorectal cancer compared to normal colonic mucosa and its expression was positively correlated with TfR1 expression. In addition, IRP2 expression was associated with mutations in BRAF. The MEK inhibitor trametinib suppressed IRP2 and this was associated with a suppression in TfR1 and the labile iron pool (LIP). Moreover, epidermal growth factor stimulation resulted in decreased ferritin expression and an increase in the LIP which were independent of IRP2. Results presented here suggest that ablating IRP2 provides a therapeutic platform for intervening in colorectal tumorigenesis.     

铁调节蛋白2在恶性肿瘤中的研究进展

铁调节蛋白2（iron regulatory protein 2,IRP2）是继IRP1后被发现,存在于细胞胞浆中的一种RNA结合蛋白,由Irp2基因编码。IRP2主要通过与铁蛋白（Ferrintin,Fn）和转铁蛋白受体（transferrin receptor,TfR）的mRNA中的铁反应元件（iron-responsive elements,IREs）相结合来调节细胞内铁的利用和储存。IPR2是维持细胞内铁稳态必不可少的调节蛋白。近年来,越来越多的研究发现,IRP2在包括结肠癌、胰腺癌、肝癌和乳腺癌等多种恶性肿瘤中具有致癌作用,可通过调节铁、激活癌基因和抑制抑癌基因等方式影响恶性肿瘤的发生、发展和预后等,可能是恶性肿瘤中一个新的潜在治疗靶点。本文就IRP2在恶性肿瘤中的研究进展进行综述。     

Altered iron metabolism,inflammation, transferrin receptors,and ferritin expression in non-small-cell lung cancer

The involvement of iron and inflammation parameters on overall survival in non-small-cell lung cancer (NSCLC) patients was studied. Furthermore, transferrin receptors 1 (TfR1) and ferritin expression in tumor tissue, tumor stroma, and normal lung tissue were analyzed. Iron metabolism and inflammation parameters were determined by automated laboratory measurements at the time of diagnosis. TfR1 and ferritin expression were determined by immuno-histochemical methods. About 50% of patients survived 12 months only. At the time of diagnosis more than half of the patients had anemia and significantly elevated serum ferritin. Iron content of serum ferritin (ICF) was below the reference values in 90% of patients. Furthermore, ICF showed positive correlation with iron metabolic parameters and survival but negative correlation with serum ferritin and ESR. The expression of TfR1 and ferritin in tumor cells was observed in 88% or 62% of patients, respectively. Tumor stroma was TfR1 negative and sporadically ferritin positive. Tumor tissue ferritin expression showed negative correlation with serum iron and hematokrit (Ht), and positive correlation with ferritin, erythrocyte sedimentation rate (ESR), α-1 globulin, and α-2 globulin. Positive correlation was found between TfR1 expression in tumor tissue and α-globulin. The correlation between TfR1/ferritin expression in tumor tissue and ICF or survival was not observed. Therefore, we conclude that elevated serum ferritin in sera of NSCLC patients is the result of inflammation and oxidative stress rather than body iron overload. Higher expression of ferritin in tumor tissue may be the consequence of iron deficiency or local toxicity induced by environmental factors.     

Iron regulatory protein 1 promotes ferroptosis by sustaining cellular iron homeostasis in melanoma

Melanoma, the most aggressive skin cancer, is mainly treated with BRAF inhibitors or immunotheareapy. However, most patients who initially responded to BRAF inhibitors or immunotheareapy become resistant following relapse. Ferroptosis is a form of regulated cell death characterized by its dependence on iron ions and the accumulation of lipid reactive oxygen species (ROS). Recent studies have demonstrated that ferroptosis is a good method for tumor treatment, and iron homeostasis is closely associated with ferroptosis. Iron regulatory protein (IRP)1 and 2 play important roles in maintaining iron homeostasis, but their functions in ferroptosis have not been investigated. The present study reported that the expression of IRP1 and IRP2 was increased by the ferroptosis inducers erastin and RSL3 in melanoma cells. Depletion of IRP1 significantly suppressed erastin- and RSL3-induced ferroptosis. IRP2 had a weak effect but could enhance the promoting function of IRP1 on ferroptosis. Further, erastin and RSL3 promoted the transition of aconitase 1 to IRP1, which regulated downstream iron metabolism proteins, including transferrin receptor (TFRC), ferroportin (FPN) and ferritin heavy chain 1 (FTH1). Moreover, overexpression of TFRC and knockdown of FPN and FTH1 significantly promoted erastin- and RSL3-induced ferroptosis in IRP1 knockdown melanoma cells. Collectively, the present findings indicate that IRP1 plays an essential role in erastin- and RSL3-induced ferroptosis by regulating iron homeostasis.     

Normal human oral keratinocytes are more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine-induced cytotoxicity and apoptosis than HPV-immortalized oral keratinocytes

Exposure of HPV-immortalized, but not normal human oral keratinocytes, to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) renders the cells tumorigenic. The underlying mechanism of this differential response of normal and immortalized cells was investigated. Normal primary human oral keratinocytes and three HPV-immortalized human oral keratinocyte cell lines exposed to MNNG were evaluated for survival rate, single and double-strand DNA breaks, and the expression of p53 and bcl-2 proteins. MNNG exposure for 2 h induced both greater cytotoxicity and a more rapid kinetic of cell death in normal keratinocytes than in the immortalized cells. Further, normal keratinocytes were more sensitive to lower concentrations of MNNG that were subtoxic for the immortalized cells. Likewise, with lower concentration of MNNG (50 mu M), significant single-strand DNA breaks in normal keratinocytes were induced whereas no such effect was seen in the immortalized cells. Double-strand DNA fragmentation (apoptosis) was observed in normal keratinocytes exposed to 50 mu M MNNG but not in the immortalized cells. Higher concentrations of MNNG (100 mu M) were toxic to both normal and immortalized cells although the normal cells were still more sensitive and with faster kinetics of cell death. MNNG-induced apoptosis was not attributable to down regulation of the anti-apoptotic product bcl-2 in normal cells; however, exposure of normal keratinocytes to MNNG did result in induction of the apoptotic gene p53. No change in p53 level was seen in the immortalized cells. These findings suggest that the selective sensitivity of normal keratinocytes to MNNG-induced apoptosis is in part due to the induction of p53. The HPV-immortalized cells are resistant to MNNG-induced apoptosis and therefore are capable of undergoing mutations affecting cell proliferation and resulting in tumori-genicity.     

Areca nut exposure increases secretion of tumor‐promoting cytokines in gingival fibroblasts that trigger DNA damage in oral keratinocytes

Molecular crosstalk between cancer cells and fibroblasts has been an emerging hot issue in understanding carcinogenesis. As oral submucous fibrosis (OSF) is an inflammatory fibrotic disease that can potentially transform into squamous cell carcinoma, OSF has been considered to be an appropriate model for studying the role of fibroblasts during early stage carcinogenesis. In this sense, this study aims at investigating whether areca nut (AN)‐exposed fibroblasts cause DNA damage of epithelial cells. For this study, immortalized hNOF (hTERT‐hNOF) was used. We found that the levels of GRO‐α, IL‐6 and IL‐8 increased in AN‐exposed fibroblasts. Cytokine secretion was reduced by antioxidants in AN‐exposed fibroblasts. Increase in DNA double strand breaks (DSB) and 8‐oxoG FITC‐conjugate was observed in immortalized human oral keratinocytes (IHOK) after the treatment of cytokines or a conditioned medium derived from AN‐exposed fibroblasts. Cytokine expression and DNA damage were also detected in OSF tissues. The DNA damage was reduced by neutralizing cytokines or antioxidant treatment. Generation of reactive oxygen species (ROS) and DNA damage response, triggered by cytokines, were abolished when NADPH oxidase (NOX) 1 and 4 were silenced in IHOK, indicating that cytokine‐triggered DNA damage was caused by ROS generation through NOX1 and NOX4. Taken together, this study provided strong evidence that blocking ROS generation might be a rewarding approach for cancer prevention and intervention in OSF.     

The Protective Effect of Antioxidants in Areca Nut Extract-Induced Oral Carcinogenesis

Objective: Oral submucous fibrosis (OSF) is the premalignant disorder associated with fibrosis and epithelial atrophy. Areca Nut (AN) is the most significant risk factors for OSF. However, the molecular mechanism behind AN induced OSF remains unclear, and there exists no effective treatment for the malignant disorder. We aimed to investigate whether AN-extract causes epithelial-mesenchymal transition (EMT) in oral keratinocytes, and evaluated the therapeutic potential of antioxidants. Methods: The HPV16 E6/E7-transfected immortalized human oral keratinocytes (IHOK) were employed in the present study. For the preparation of AN-extract, dried AN was dissolved in distilled water overnight. The solution was centrifuged and the supernatant was collected for further use. For the determination of change in cytokine levels, ELISA was performed. To investigate EMT-related protein expression and phenotype, immunoblot and immunofluorescence were performed. Results: Among tumor-promoting cytokines (Gro-α, IL-6 and IL-8), IL-6 was remarkably increased by AN in IHOK. AN-extract induced EMT phenotypes, such as cell elongation, up-regulation of vimentin and snail. After treatment with neutralizing antibody of IL-6, AN-induced snail expression was reduced remarkably. Collectively, AN-extract induced IL-6 expression and mediated EMT. The use of antioxidants (EGCG, glutathione and NAC) significantly reduced IL-6 expression in AN-treated IHOK. Also, AN-decreased E-cadherin and increased vimentin were reversed by antioxidants, indicating that the effectiveness of antioxidants in inhibiting IL-6-induced EMT by AN. Conclusion: AN promotes EMT and antioxidants interrupt AN-induced-EMT in oral keratinocytes. Consequently, it is proposed that antioxidants could prevent AN-induced carcinogenesis and function as a prototype for developing therapeutic interventions of OSF.     

Genome-wide analysis of oral cancer--early results from the Cancer Genome Anatomy Project

The Cancer Genome Anatomy Project (CGAP) is a large cooperative effort sponsored by the US National Institutes of Health designed to find, catalog and annotate genes that are expressed during cancer development. In the past 2 years, the CGAP has sequenced over 700,000 clones from approximately 140 cDNA libraries, resulting in the identification of over 30,000 new human genes. As a first step in applying this project to oral cancer we entered four cell lines--two from oral cancer, one from primary oral keratinocytes, and one from oral keratinocytes which had been immortalized by human papillomavirus. Libraries of cDNA were made and sequenced and the data were deposited in GenBank. The expressed genes were then identified where possible. The cell lines, and the total number of expressed genes that were cloned from each were: HN3 (oral cancer), 263 genes; HN4 (oral cancer), 550 genes; HN5 (primary keratinocytes), 237 genes; HN6 (immortalized keratinocytes), 408 genes. The total number of different genes that were found was 1160. A total of 38 new genes, of unknown function, were discovered. The data presented here represent a beginning of the application of the CGAP technology to oral cancer. Even though the data are still quite incomplete, they already represent a large quantity of new information and clones of potential utility to the oral cancer community, and provide a glimpse of the data sets to be forthcoming from the Project. It must therefore be expected that there will soon be a large expansion in the volume of data regarding the genetics of oral cancer. Those who study this disease must be prepared to develop new methods of analysis and storage for handling the oncoming volumes of information.     

Doxorubicin irreversibly inactivates iron regulatory proteins 1 and 2 in cardiomyocytes: evidence for distinct metabolic pathways and implications for iron-mediated cardiotoxicity of antitumor therapy.

Changes in iron homeostasis have been implicated in cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX). Certain products of DOX metabolism, like the secondary alcohol doxorubicinol (DOXol) or reactive oxygen species (ROS), may contribute to cardiotoxicity by inactivating iron regulatory proteins (IRP) that modulate the fate of mRNAs for transferrin receptor and ferritin. It is important to know whether DOXol and ROS act by independent or combined mechanisms. Therefore, we monitored IRP activities in H9c2 rat embryo cardiomyocytes exposed to DOX or to analogues which were selected to achieve a higher formation of secondary alcohol metabolite (daunorubicin), a concomitant increase of alcohol metabolite and decrease of ROS (5-iminodaunorubicin), or a defective conversion to alcohol metabolite (mitoxantrone). On the basis of such multiple comparisons, we characterized that DOXol was able to remove iron from the catalytic Fe-S cluster of cytoplasmic aconitase, making this enzyme switch to the cluster-free IRP-1. ROS were not involved in this step, but they converted the IRP-1 produced by DOXol into a null protein which did not bind to mRNA, nor was it able to switch back to aconitase. DOX was also shown to inactivate IRP-2, which does not assemble or disassemble a Fe-S cluster. Comparisons between DOX and the analogues revealed that IRP-2 was inactivated only by ROS. Thus, DOX can inactivate both IRP through a sequential action of DOXol and ROS on IRP-1 or an independent action of ROS on IRP-2. This information serves guidelines for designing anthracyclines that spare iron homeostasis and induce less severe cardiotoxicity.     

Altered iron metabolism, transferrin receptor 1 and ferritin in patients with colon cancer

In this study, the level and distribution of transferrin receptor 1 (TfR1) and ferritin in colorectal carcinoma and in normal colon epithelium has been determined relative to the tumor stage and iron status of patients using immunohistochemical staining methods. While the majority of carcinoma patients were anemic, no relationship between the level of colon tissue ferritin and TfR1 and the systemic parameters of iron metabolism was evident. Furthermore, no association between ferritin content and the grade of colorectal carcinoma was observed. However, a relationship between the expression of TfR1 and the grade of colorectal carcinoma was observed. In this case high expression of TfR1 was found in colorectal carcinoma samples of Dukes A or B grade, and well differentiated colorectal carcinoma cells. In comparison, weak or no expression of TfR1 was observed in carcinoma samples of Dukes C or D grade with poorly differentiated cells and in carcinoma samples that had lymph node infiltration and distant metastasis.     

Uptake of anti-anemic substance ferric-sorbitol-citrate by normal and malignant cells and its effects on expression of transferrin receptor 1 and ferritin

Iron-containing antianemic drug ferric-sorbitol-citrate (FSC) inhibits the proliferation of various cancer cell lines in vitro and causes a regression of experimental murine tumors in vivo but does not affect the proliferation of nonmalignant cells. Growth modification caused by FSC iron involves a diminished expression of Bcl-2 and an overexpression of p53 proto-oncogene, accompanied by an increased incidence of apoptosis. Aiming to evaluate further the activity principle of the anticancer effects of this antianemic drug, in this study, we analyzed the utilization of iron from FSC and the effects of FSC iron on transferrin receptor 1 (TfR1) and ferritin expression. Without FSC iron, all the cell lines had an equal expression of TfR1, but if cultured in FSC-supplemented medium, human colon SW620 and laryngeal carcinoma Hep cells exhibited a lower expression of TfR1-positive cells than nonmalignant Wi38 fibroblasts and pancreatic carcinoma MiaPaCa2 cells. The most sensitive to FSC iron were colon carcinoma SW620 cells, whereas Wi38 fibroblasts were not sensitive at all. Increased iron uptake by colon carcinoma cells was noticed in the first 3 hours of the incubation with FSC iron, whereas higher FSC iron concentrations and longer incubation also impaired ferritin expression in SW260 colon carcinoma cells. Thus, the anticancer ability of FSC could result from its higher initial utilization of iron and consecutive negative signal for the expression of TfR1 in tumor cells. Tumor cells containing lower amounts of ferritin are probably more sensitive to oxidative stress caused by iron overload, whereas FSC iron itself was proven to be chemically stable and did not induce lipid peroxidation.     

The protein degradation system involved in the regulation of iron metabolism

Iron is an essential nutrient for almost all organisms, but at the same time excess iron is toxic to generate radicals and damages important macromolecules. Thus, organisms have developed a strict regulatory system for iron metabolism. We have studied the iron-mediated regulatory mechanism of a regulator of the iron metabolism, IRP2, and found that IRP2 is regulated by an oxidation-induced degradation, which is triggered by heme and molecular oxygen. Recent advances in the analyses of iron regulation in cells and animals reveal that the protein degradation system is involved in the iron-mediated regulation of another regulator of iron metabolism, IRP1. Considering that heme is generated in the matrix of mitochondria, our observation that IRP2 is regulated by the protein degradation pathway triggered by the heme-mediated oxidation indicates the involvement and importance of mitochondria in cellular iron sensing as well as the regulation of the iron metabolism.     

Dose and interval relationship for the interaction of WR-2721 and nitrogen mustard with normal and malignant cells

The protective effect of WR-2721 on nitrogen mustard (HN2) cytotoxicity against hematopoietic stem cells as well as the potentiation effect previously noted against leukemia cells was further examined for dose and interval dependency for these opposite effects. For AKR leukemia cells, a dose dependent potentiation of HN2 cytotoxicity was noted with WR-2721. The magnitude of the potentiation was about 10-fold with 5 mg/mouse WR-2721 and nearly 100-fold at 10 mg/mouse. Interval studies showed maximum potentiation occurred when WR-2721 was given close in time to HN2 and decreased in magnitude as the interval between the agents increased. However, a pronounced potentiation of cytotoxicity was also found when HN2 followed WR-2721 by 4 to 12 hr. For hematopoietic stem cells, a protective effect of WR-2721 on HN2 cytotoxicity was observed. This effect decreased rapidly with interval between the agents with no protection noted for an interval of 6 hr. These results indicate that agents like WR-2721 may have an important therapeutic role in chemotherapy.     

Potentiation of nitrogen mustard cytotoxicity by disulfiram, diethyldithiocarbamic acid, and diethylamine in mice

Disulfiram (DSF) and its metabolites, diethyldithiocarbamic acid (DDC) and diethylamine (DEA), were studied as pretreatments in combination with the alkylating agent nitrogen mustard (HN2) on the cytotoxicity of HN2 against both leukemia cells and normal hematopoietic stem cells. Both time intervals and dose relationships were examined. DSF showed a substantial potentiation of HN2 cytotoxicity against murine AKR leukemia cell spleen colony-forming units (LCFU) when given i.p. between 15 to 30 min prior to HN2 i.v. treatment. For 3 mg DSF/mouse pretreatment, leukemia LCFU survival was about 10(-6) whereas it was about 10(-2) for HN2 alone. The extent of this potentiation decreased as the time between treatments increased. Significant potentiation was noted even when a low dose of DSF (0.25 mg/mouse) was administered 15 min before HN2. However, DSF had little if any effect on the modulation of HN2 cytotoxicity to normal hematopoietic cell spleen colony-forming units (NCFU). DDC showed an increasing potentiation of HN2 cytotoxicity against LCFU when given i.p. prior to HN2 i.v. treatment. The maximum effect was noted between 2 and 4 h with a surviving fraction for LCFU between 10(-5) and 10(-6) for 20 mg/mouse DDC pretreatment. The extent of this effect then decreased as the time interval increased beyond 4 h, but it was still significant for the 24-h interval. This pronounced potentiation effect was dose dependent for DDC. The compound exhibited a protective effect against HN2 cytotoxicity to NCFU when given 15 min before HN2. This protection decreased with increased time interval. DEA (20 mg/mouse) did not show a significant potentiation of HN2 cytotoxicity against LCFU when administered i.p. prior to HN2. Also, DEA did not show any significant protection of NCFU.     

Perifosine, a novel alkylphospholipid, induces p21(WAF1) expression in squamous carcinoma cells through a p53-independent pathway, leading to loss in cyclin-dependent kinase activity and cell cycle arrest

Alkylphospholipids (ALKs) are a novel class of antineoplastic compounds that display potent antiproliferative activity against several in vitro and in vivo human tumor models. However, the mechanism by which these agents exert this desired effect is still unclear. In this study, we investigated the effect of perifosine, a p.o.-bioavailable ALK, on the cell cycle kinetics of immortalized keratinocytes (HaCaT) as well as head and neck squamous carcinoma cells. All cells were sensitive to the antiproliferative properties of perifosine with an IC(50) of similar0.6-8.9 microM. Cell cycle arrest at the G(1)-S and G(2)-M boundaries was observed in HN12, HN30, and HaCaT cells independent of p53 function, and this effect was preceded by loss in cdc2 and cyclin-dependent kinase (cdk) 2 activity. Analysis of cdk complexes in vitro demonstrated that perifosine, up to 20 microM, did not directly interfere with these enzymes. However, aphidicolin-synchronized HN12 cells released in the presence of perifosine (10 microM) demonstrated increased expression of total p21(WAF1) and increased association of p21(WAF1) with cyclin-cdk complexes resulting in reduced cdc2 activity. HCT116 isogenic cell lines were used to assess the role of p21(WAF1) induction by perifosine. This compound (20 microM) induced both G(1)-S and G(2)-M cell cycle arrest, together with p21(WAF1) expression in both p53 wild-type and p53(-/-) clones. By contrast, p21(-/-) variants demonstrated no p21(WAF1) induction or cell cycle arrest. Similar results were obtained with other ALK congeners (miltefosine and edelfosine). These data, therefore, indicate that perifosine blocks cell cycle progression of head and neck squamous carcinoma cells at G(1)-S and G(2)-M by inducing p21(WAF1), irrespective of p53 function, and may be exploited clinically because the majority of human malignancies harbor p53 mutations.     

Metastasis-associated protein (MTA)1 enhances migration, invasion, and anchorage-independent survival of immortalized human keratinocytes

The human metastasis-associated gene (MTA1), a member of the nucleosome remodeling complex with histone deacetylase activity, is frequently overexpressed in biologically aggressive epithelial neoplasms. Here, we extend this observation to squamous carcinoma cells, which express high levels of MTA1 relative to normal or immortalized keratinocytes. To address functional aspects of MTA1 expression, we established variants of human immortalized keratinocytes (HaCaT cells) by expressing MTA1 cDNA in both the sense and antisense orientations. We demonstrate that (1) forced MTA1 expression enhances migration and invasion of immortalized keratinocytes; (2) MTA1 expression is necessary but not sufficient for cell survival in the anchorage independent state; (3) MTA1 contributes to expression of the anti-apoptotic Bcl-2 family member Bcl-x(L); (4) MTA1 expression in immortalized keratinocytes depends, in part, on activation of the epidermal growth factor receptor (EGFR). These results establish that, in keratinocytes, MTA1 expression contributes to several aspects of the metastatic phenotype including survival in the anchorage independent state, migration, and invasion.     

The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels

Inactivation of the TCA cycle enzyme, fumarate hydratase (FH), drives a metabolic shift to aerobic glycolysis in FH-deficient kidney tumors and cell lines from patients with hereditary leiomyomatosis renal cell cancer (HLRCC), resulting in decreased levels of AMP-activated kinase (AMPK) and p53 tumor suppressor, and activation of the anabolic factors, acetyl-CoA carboxylase and ribosomal protein S6. Reduced AMPK levels lead to diminished expression of the DMT1 iron transporter, and the resulting cytosolic iron deficiency activates the iron regulatory proteins, IRP1 and IRP2, and increases expression of the hypoxia inducible factor HIF-1α, but not HIF-2α. Silencing of HIF-1α or activation of AMPK diminishes invasive activities, indicating that alterations of HIF-1α and AMPK contribute to the oncogenic growth of FH-deficient cells.