The role of ZIP8 down‐regulation in cadmium‐resistant metallothionein‐null cells

The mechanisms of cellular cadmium uptake in mammalian cells remain obscure. To solve this problem, we established cadmium‐resistant cells (A7 and B5) from metallothionein‐null mouse cells, and found that cadmium accumulation was markedly suppressed in these cells. DNA microarray and real‐time PCR analyses revealed that expressions of ZIP (Zrt‐, Irt‐related protein) 8 and ZIP14 were down‐regulated in A7 and B5 cells. In particular, both mRNA and protein levels of ZIP8 were markedly suppressed in A7 and B5 cells. Introduction of short hairpin RNA (shRNA) of ZIP8 into parental cells reduced the accumulation of cadmium to about 35% of that of mock‐transfected cells, whereas the introduction of shRNA of divalent metal transporter 1 hardly changed cadmium accumulation. Thus, the cadmium resistance in A7 and B5 cells may be conferred primarily by the down‐regulation of ZIP8. In mouse tissues, high expression of ZIP8 was noted in the liver, kidney, lung and testis. These data suggest that ZIP8 plays an important role in cellular uptake of cadmium. Copyright © 2009 John Wiley & Sons, Ltd.     

Cross-resistance of cadmium-resistant cells to manganese is associated with reduced accumulation of both cadmium and manganese

The mechanism of cellular entry of cadmium remains unclear. We have previously established cadmium-resistant cells from mouse embryonic cells of metallothionein (MT)-null mice, and demonstrated that the down-regulation of a zinc transporter, Zrt/Irt-related protein (ZIP) 8, was responsible for the reduced cadmium incorporation into cells. In the present study, we developed cadmium-resistant cells (A+70 and B+70) from mouse embryonic cells of MT-expressing wild-type mice. The LC?? values of CdCl? for A+70 and B+70 cells were about 200 μM while that of the parental cells was 30 μM. We found that the cadmium resistance of these cells was conferred not only by enhanced expression of MT, but also by a decrease in cadmium accumulation. Since the uptake rates of cadmium into A+70 and B+70 cells were lowered, we determined the expression levels of the metal transporters and channels potentially involved in the cellular uptake of cadmium. We found a down-regulation of multiple transport systems, including ZIP8, divalent metal transporter 1 (DMT1), and α? subunits of L-type (Ca(V)1.2) and T-type (Ca(V)3.1) voltage-dependent calcium channels, in A+70 and B+70 cells. Furthermore, A+70 and B+70 cells exhibited cross-resistance to cytotoxicity of MnCl?, probably due to a marked decrease in manganese uptake in these cells. These results suggest that the suppressed expression of ZIP8 and DMT1, which are known to have affinities for both cadmium and manganese, may be responsible for the reduction in the uptake, and consequently the cytotoxicity, of cadmium and manganese in A+70 and B+70 cells.     

Discovery of ZIP transporters that participate in cadmium damage to testis and kidney

It has been known for decades that cadmium (Cd) must enter the cell to cause damage, but there was no mechanism to explain genetic differences in response to Cd toxicity until 2005. Starting with the mouse Cdm locus associated with differences in Cd-induced testicular necrosis between inbred strains, a 24.6-centiMorgan region on chromosome 3 was reduced ultimately to 880 kb; in this segment is the Slc39a8 gene encoding the ZIP8 Zn²⁺/HCO₃⁻ symporter. In endothelial cells of the testis vasculature, Cd-sensitive mice exhibit high ZIP8 expression, Cd-resistant mice exhibit very low expression. A 168.7-kb bacterial artificial chromosome (BAC) from a 129S6 (Cd-sensitive) BAC library containing the Slc39a8 gene was inserted into the Cd-resistant C57BL/6J genome: Cd treatment produced testicular necrosis in BAC-transgenic BTZIP8-3 mice but not in non-transgenic littermates, thereby proving that the Slc39a8 gene is indeed the Cdm locus. Cd-induced renal failure also occurred in these BTZIP8-3 mice. Immunohistochemistry showed highly expressed ZIP8 protein in the renal proximal tubular epithelial apical surface, suggesting that ZIP8 participates in Cd-induced renal failure. Slc39a14, most closely evolutionarily related to Slc39a8, encodes differentially-spliced products ZIP14A and ZIP14B that display properties similar to ZIP8. ZIP8 in alveolar cells brings environmental Cd into the organism and ZIP14 in intestinal enterocytes carries Cd into the organism and into the hepatocyte. We believe these two transporters function endogenously as Zn²⁺/HCO₃⁻ symporters important in combating inflammation and carrying out other physiological functions; Cd is able to displace the endogenous cation, enter the cell, and produce tissue damage and disease.     

异硫氰酸苯已酯诱导骨髓瘤U266细胞P16基因去甲基化

目的研究异硫氰酸苯己酯(PHI)对骨髓瘤U266细胞株P16基因的去甲基化作用并探讨其作用机制。方法应用半定量逆转录-聚合酶链反应检测U266细胞经过PHI处理后DNA甲基转移酶DNMT1、DNMT3a和DNMT3b基因的mRNA的表达变化,用甲基化特异性聚合酶链反应检测PHI作用前后U266细胞株P16基因甲基化状态的变化。结果使用不同浓度的PHI处理U266细胞72h后,U266细胞表达DNMT1、DNMT3a、DNMT3bmRNA的水平明显降低并呈浓度依赖性。以不同浓度的PHI处理U266细胞10d后,P16基因的甲基化状态被逐渐逆转。结论 PHI可能通过抑制DNA甲基转移酶的表达水平诱导P16基因产生去甲基化,使失活的抑癌基因重新激活,诱导瘤细胞凋亡。     

Dust fall PM2.5-induced lung inflammation in rats is associated with hypermethylation of the IFN-γ gene promoter via the PI3K-Akt-DNMT3b pathway

Inflammation is one of the major adverse effects of fine particulate matter (PM_2.5) on the lung system; however, its mechanisms remain unclear. Rats were exposed to different concentrations of PM_2.5 to investigate the mechanism of short-term exposure-induced lung inflammation. The regulation of PI3K-Akt and DNA methyltransferase 3b (DNMT3b) was assessed by using a PI3K inhibitor and a DNA methyltransferase inhibitor. We found that PM_2.5 could decrease interferon-γ (IFN-γ) levels and increase interleukin 4 (IL-4), IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF) to promote eosinophil infiltration and eventually lead to allergic pulmonary inflammation. Moreover, the CpG island methylation rate of the IFN-γ promoter and the protein expression of DNMT3b, PI3K and p-Akt were increased in lung tissues after PM_2.5 exposure. Both inhibitors reversed the CpG island hypermethylation of IFN-γ. In conclusion, in PM_2.5-induced lung injury, the activated PI3K-Akt pathway, via an increase in DNMT3b expression, is involved in CpG hypermethylation of the IFN-γ gene promoter.     

ZIP8, member of the solute-carrier-39 (SLC39) metal-transporter family: characterization of transporter properties

Cadmium is a dangerous metal distributed widely in the environment. Members of our laboratory recently identified the ZIP8 transporter protein, encoded by the mouse Slc39a8 gene, to be responsible for genetic differences in response to cadmium damage of the testis. Stable retroviral infection of the ZIP8 cDNA in mouse fetal fibroblast cultures (rvZIP8 cells) leads to as much as a 10-fold increase in the rate of intracellular cadmium influx and accumulation. In the present study, we showed that cadmium uptake operated maximally at pH 7.5 and a temperature of 37 degrees C and was inhibited by cyanide. Of more than a dozen cations tested, manganese(II) was the best competitive cation for cadmium uptake. The Km for Cd2+ uptake was 0.62 microM, and the Km for Mn2+ uptake was 2.2 microM; thus, manganese is probably the physiological substrate for ZIP8. Cadmium uptake was independent of sodium, potassium or chloride ions, but strongly dependent on the presence of bicarbonate. By Western blot analysis of rvZIP8 cells, we showed that ZIP8 protein was glycosylated. Using Z-stack confocal microscopy in Madin-Darby canine kidney polarized epithelial cells, we found that ZIP8 was localized on the apical side-implying an important role for manganese or cadmium uptake and disposition. It is likely that ZIP8 is a Mn2+/HCO3- symporter, that a HCO3- gradient across the plasma membrane acts as the driving force for manganese uptake, and that cadmium is a rogue hitchhiker displacing manganese to cause cadmium-associated disease.     

Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16^INK4a and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans.     

5-氮-2′脱氧胞苷对T24膀胱癌细胞RUNX3基因去甲基化的转录调节作用

目的研究甲基化抑制剂5-氮-2′-脱氧胞苷(5-Aza-dc)对T24膀胱癌细胞生长的抑制作用及对RUNX3基因的转录调节作用。方法不同浓度5-Aza-dc处理T24膀胱癌细胞后,采用四唑盐(MTT)比色观察细胞经药物处理前后的生长活性;以半定量反转录-聚合酶链反应(RT-PCR),甲基化特异性PCR(methylationspecific PCR,MSP)检测细胞处理前后RUNX3基因mRNA的表达,RUNX3基因甲基化状态;应用流式细胞仪进行细胞凋亡率的检测。结果5-Aza-dc抑制T24膀胱癌细胞的生长;细胞凋亡增加;RUNX3基因甲基化逆转;mRNA表达恢复。以上作用与药物存在剂量依赖性。结论5-Aza-dc能逆转T24膀胱癌细胞的RUNX3基因甲基化,恢复该基因的表达,诱导T24膀胱癌细胞凋亡。     

Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter

The mouse and human genomes contain 14 highly conserved SLC39 genes. Viewed from an evolutionary perspective, SLC39A14 and SLC39A8 are the most closely related, each having three noncoding exons 1. However, SLC39A14 has two exons 4, giving rise to Zrt- and Irt-related protein (ZIP)ZIP14A and ZIP14B alternatively spliced products. C57BL/6J mouse ZIP14A expression is highest in liver, duodenum, kidney, and testis; ZIP14B expression is highest in liver, duodenum, brain, and testis; and ZIP8 is highest in lung, testis, and kidney. We studied ZIP14 stably retroviral-infected mouse fetal fibroblast cultures and transiently transfected Madin-Darby canine kidney (MDCK) polarized epithelial cells. Our findings include: 1) ZIP14-mediated cadmium uptake is proportional to cell toxicity, but manganese is not; 2) ZIP14B has a higher affinity than ZIP14A toward Cd(2+) (K(m) = 0.14 versus 1.1 microM) and Mn(2+) uptake (K(m) = 4.4 versus 18.2 microM); 3) ZIP14A- and ZIP14B-mediated Cd(2+) uptake is most inhibited by Zn(2+), and next by Mn(2+) and Cu(2+); 4) like ZIP8, ZIP14A- and ZIP14B-mediated Cd(2+) uptake is dependent on extracellular HCO(3)(-); 5) like ZIP8, ZIP14 transporters are localized on the apical surface of MDCK-ZIP cells; and 6) like ZIP8, ZIP14 proteins are glycosylated. Tissues such as intestine and liver, located between the environment and the animal, show high levels of ZIP14; given the high affinity for ZIP14, Cd(2+) is likely to act as a rogue hitchhiker-displacing Zn(2+) or Mn(2+) and entering the body to cause unwanted cell damage and disease.     

DNA methylation and cancer therapy

Vertebrate DNA is modified by methyl moieties at the 5'-position of cytosine rings residing in the di-nucleotide sequence CpG. Approximately 80% of CpG dinucleotide sequences are methylated. The pattern of distribution of methylated CGs is cell-type specific and correlates with gene expression programming and chromatin structure. Three kinds of seemingly contradictory aberrations in DNA methylation are observed in cancer, global hypomethylation, and regional hypermethylation and deregulated level of expression of DNA methyltransferases. It was previously proposed that the DNA methylation machinery is a candidate target for anticancer therapy. Inhibition of hypermethylation was the first therapeutic target. However, recent data suggests that inhibition of DNA methylation might have untoward effects such as induction of genes involved in metastasis. This review discusses the relative role of the three levels of alteration in the DNA methylation in cancer, proposes a unified hypothesis on the relative roles of increased DNA methyltransferase as well as the coexistence of hypo -and hyper- methylation in cancer and its possible implications on anticancer therapy.     

5-氮杂-2′-脱氧胞苷诱导卵巢癌细胞系p16基因去甲基化及其表达

目的观察去甲基化制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对体外培养的卵巢癌细胞CAOV3p16基因启动子区甲基化状态及表达的影响,探讨卵巢癌细胞p16基因失活的机制及去甲基化制剂对p16基因表达的调控。方法应用不同浓度的5-Aza-CdR(1×10-7mol/L,5×10-7mol/L,1×10-6mol/L,5×10-6mol/L)处理体外培养的卵巢癌细胞CAOV3后,甲基化特异性聚合酶链反应(PCR)(MSP)法检测用药前后细胞中p16基因的甲基化状态,RT-PCR法及Western-blot法检测用药前后细胞中p16 mRNA及蛋白表达的变化。结果p16基因在卵巢癌细胞系CAOV3中启动子区呈异常甲基化状态,在 mRNA及蛋白水平低表达。经过5-Aza-CdR处理后,p16基因启动子区呈去甲基化状态,其 mRNA及蛋白表达显著增强(P<0.01)。结论启动子区异常甲基化是卵巢癌细胞p16基因失活的主要原因之一,去甲基化制剂-5-Aza-CdR能逆转p16基因甲基化状态,从而调控p16基因表达。     

Epigenetic CpG Demethylation of the Promoter and Reactivation of the Expression of Neurog1 by Curcumin in Prostate LNCaP Cells

Curcumin (CUR), a major bioactive polyphenolic component from turmeric curry, Curcuma longa, has been shown to be a potent anti-cancer phytochemical with well-established anti-inflammatory and anti-oxidative stress effects. Chromatin remodeling-related epigenetic regulation has emerged as an important mechanism of carcinogenesis, chemoprevention, and chemotherapy. CUR has been found to inhibit histone acetyltransferase activity, and it was also postulated to be a potential DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitor. In this study, we show that when human prostate LNCaP cells were treated with CUR, it led to demethylation of the first 14 CpG sites of the CpG island of the Neurog1 gene and restored the expression of this cancer-related CpG-methylation epigenome marker gene. At the protein level, CUR treatment had limited effects on the expression of epigenetic modifying proteins MBD2, MeCP2, DNMT1, and DNMT3a. Using ChIP assay, CUR decreased MeCP2 binding to the promoter of Neurog1 dramatically. CUR treatment showed different effects on the protein expression of HDACs, increasing the expression of HDAC1, 4, 5, and 8 but decreasing HDAC3. However, the total HDAC activity was decreased upon CUR treatment. Further analysis of the tri-methylation of histone 3 at lysine 27 (H3K27me3) showed that CUR decreased the enrichment of H3K27me3 at the Neurog1 promoter region as well as at the global level. Taken together, our present study provides evidence on the CpG demethylation ability of CUR on Neurog1 while activating its expression, suggesting a potential epigenetic modifying role for this phytochemical compound in human prostate cancer cells.     

5-氮-2’脱氧胞苷诱导肺癌细胞系CDH13基因去甲基化及其表达

目的 观察去甲基化制剂5-氮-2’脱氧胞苷(5-aza-2’-deoxycytidine,5-Aza-dC)对人肺癌细胞系A549和LTEP-a2中CDH13基因启动子区甲基化状态及表达的影响,探讨肺癌细胞CDH13基因失活的机制及去甲基化制剂对CDH13基因表达的调控作用. 方法 5-Aza-dC处理体外培养的肺癌细胞A549、LTEP-a2及正常肺细胞系HFL1后,用甲基化特异性 PCR( MSP) 法检测处理前后细胞中CDH13基因的甲基化状态,半定量逆转录 聚合酶链反应(RT PCR)法检测用药前后细胞中CDH13 mRNA表达的变化. 结果 正常肺细胞中未见CDH13启动子甲基化,肺癌细胞A549、LTEP a2均发现CDH13启动子甲基化现象;应用 5-Aza-dC能使 CDH13基因启动子区 CpG 岛发生去甲基化. 肺癌细胞A549、LTEP-a2均可见CDH13 mRNA表达,但与正常肺细胞比较表达较弱,经药物处理癌细胞后其CDH13 mRNA的表达较前明显增强. 结论 启动子区异常甲基化是肺癌细胞CDH13基因失活的主要原因之一,去甲基化制剂5-Aza-dC能完全逆转CDH13基因甲基化状态,从而调控CDH13基因表达.     

5-Aza-2'-deoxycytidine suppresses human renal carcinoma cell growth in a xenograft model via up-regulation of the connexin 32 gene

BACKGROUND AND PURPOSE: The connexin (Cx) 32 gene, a member of the gap junction gene family, acts as a tumour suppressor gene in human renal cell carcinoma (RCC) and is down-regulated by the hypermethylation of CpG islands in a promoter region of the Cx gene. The current study investigated whether the restoration of Cx32 silenced by hypermethylation in RCC by a DNA demethylating agent could be an effective treatment against RCC. EXPERIMENTAL APPROACH: Using nude mice bearing Caki-1 cells (a human metastatic RCC cell line), the effects of 5-aza-2'-deoxycytidine (5-aza-CdR), a DNA demethylase inhibitor, on Cx32 mRNA expression and tumour growth were examined by RT-PCR, and by measuring tumour weight and volume. Cx32 expression in Caki-1 tumours was inhibited by Cx32 short interfering (si) RNA, and the effect of siRNA on 5-aza-CdR-dependent suppression of tumour growth in nude mice was evaluated. KEY RESULTS: 5-aza-CdR treatment inhibited the growth of Caki-1 cells in nude mice by 70% and increased 7-fold the level of Cx32 mRNA. The intratumour injection of Cx32 siRNA almost totally inhibited the expression of Cx32 mRNA and significantly reduced the suppression of tumour growth in 5-aza-CdR-treated nude mice. CONCLUSIONS AND IMPLICATIONS: 5-aza-CdR suppressed the growth of Caki-1 tumours in a xenograft model, by restoring Cx32 expression. This finding suggests that treatment with 5-aza-CdR could be a new effective therapy against human metastatic RCC and that Cx32 could be a potential target for the treatment of RCC.