The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo

DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 μM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.     

Antileukemic activity of combined epigenetic agents, DNMT inhibitors zebularine and RG108 with HDAC inhibitors, against promyelocytic leukemia HL-60 cells

DNMT inhibitors are promising new drugs for cancer therapies. In this study, we have observed the antileukemic action of two diverse DNMT inhibitors, the nucleoside agent zebularine and the non-nucleoside agent RG108, in human promyelocytic leukemia (PML) HL-60 cells. Zebularine but not RG108 caused dose- and time-dependent cell growth inhibition and induction of apoptosis. However, co-treatment with either drug at a non-toxic dose and all trans retinoic acid (RA) reinforced differentiation to granulocytes, while 24 or 48 h-pretreatment with zebularine or RG108 followed by RA alone or in the presence of HDAC inhibitors (sodium phenyl butyrate or BML-210) significantly accelerated and enhanced cell maturation to granulocytes. This occurs in parallel with the expression of a surface biomarker, CD11b, and early changes in histone H4 acetylation and histone H3K4me3 methylation. The application of both drugs to HL-60 cells in continuous or sequential fashion decreased DNMT1 expression, and induced E-cadherin promoter demethylation and reactivation at both the mRNA and the protein levels in association with the induction of granulocytic differentiation. The results confirmed the utility of zebularine and RG108 in combinations with RA and HDAC inhibitors to reinforce differentiation effects in promyelocytic leukemia.     

Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination

Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It’s well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR.     

Zebularine inhibits the growth of HeLa cervical cancer cells via cell cycle arrest and caspase-dependent apoptosis

Zebularine (Zeb) as a DNA methyltrasferase (DNMT) inhibitor has various cellular effects such as cell growth inhibition and apoptosis. In the present study, we evaluated the effects of Zeb on the growth and death of HeLa cervical cancer cells. Zeb inhibited the growth of HeLa cells with an IC(50) of approximately 130?μM at 72?h in a dose-dependent manner. DNA flow cytometric analysis indicated that Zeb induced an S phase arrest of the cell cycle, which was accompanied by the increased levels of cdk2 and cyclin A proteins. This agent also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (Ψ(m)), PARP-1 cleavage and the activation of caspase-3, -8 and -9. All of the tested caspase inhibitors significantly rescued some cells from Zeb-induced HeLa cell death. In relation to reactive oxygen species (ROS) and glutathione (GSH) levels, O (2) (?-) level was significantly increased in 100?μM Zeb-treated HeLa cells and caspase inhibitors reduced O (2) (?-) level in these cells. Zeb induced GSH depletion in HeLa cells, which was attenuated by caspase inhibitors. In conclusion, this is the first report that Zeb inhibited the growth of HeLa cells via cell cycle arrest and apoptosis.     

Folic Acid Acts Through DNA Methyltransferases to Induce the Differentiation of Neural Stem Cells into Neurons

The present study investigated the roles of folic acid and DNA methyltransferases (DNMTs) in the differentiation of neural stem cells (NSCs). Neonatal rat NSCs were grown in suspended neurosphere cultures and identified by their expression of SOX2 protein and capacity for self-renewal. Then NSCs were assigned to five treatment groups for cell differentiation: control (folic acid-free differentiation medium), low folic acid (8 μg/mL), high folic acid (32 μg/mL), low folic acid and DNMT inhibitor zebularine (8 μg/mL folic acid and 150 nmol/mL zebularine), and high folic acid and zebularine (32 μg/mL folic acid and 150 nmol/mL zebularine). After 6 days of cell differentiation, immunocytochemistry and western blot analyses were performed to identify neurons by β-tubulin III protein expression and astrocytes by GFAP expression. We observed that folic acid increased DNMT activity which may be regulated by the cellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and the abundance of neurons but decreased the number of astrocytes. Zebularine blocked these effects of folic acid. In conclusion, folic acid acts through elevation of DNMT activity to increase neuronal differentiation and decrease astrocytic differentiation in NSCs.     

DNA Methyltransferase Inhibitor Zebularine Inhibits Human Hepatic Carcinoma Cells Proliferation and Induces Apoptosis

Hepatocellular carcinoma is one of the most common cancers worldwide. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-_D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on hepatocellular carcinoma cell line HepG2. We demonstrate that zebularine exhibits antitumor activity on HepG2 cells by inhibiting cell proliferation and inducing apoptosis, however, it has little effect on DNA methylation in HepG2 cells. On the other hand, zebularine treatment downregulated CDK2 and the phosphorylation of retinoblastoma protein (Rb), and upregulated p21^WAF/CIP1 and p53. We also found that zebularine treatment upregulated the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). These results suggest that the p44/42 MAPK pathway plays a role in zebularine-induced cell-cycle arrest by regulating the activity of p21^WAF/CIP1 and Rb. Furthermore, although the proapoptotic protein Bax levels were not affected, the antiapoptotic protein Bcl-2 level was downregulated with zebularine treatment. In addition, the data in the present study indicate that inhibition of the double-stranded RNA-dependent protein kinase (PKR) is involved in inducing apoptosis with zebularine. These results suggest a novel mechanism of zebularine-induced cell growth arrest and apoptosis via a DNA methylation-independent pathway in hepatocellular carcinoma.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.     

Involvement of autophagy in recombinant human arginase-induced cell apoptosis and growth inhibition of malignant melanoma cells

Recombinant human arginase (rhArg) has been developed for arginine derivation therapy of cancer and is currently in clinical trials for a variety of malignant solid tumors. In this study, we reported for the first time that rhArg could induce obvious autophagy in human melanoma cells; inhibition of autophagy by chloroquine (CQ) significantly increased rhArg-induced cell apoptosis and growth inhibition of A375 cells. A significant increase in mitochondrial membrane potential loss and elevated intracellular reactive oxygen species (ROS) levels were detected in A375 cells after rhArg treatment when compared with control. Membrane transition inhibitor cyclosporine A blocked autophagy and accelerated cell death induced by rhArg, indicating that rhArg induced autophagy via mitochondria pathway. Furthermore, antioxidant N-acetyl-l-cysteine suppressed rhArg-induced autophagy and rescued cells from cell growth inhibition, suggesting that ROS played an important role in rhArg-induced A375 cell growth inhibition and autophagy. Akt/mTOR signaling pathway was involved in autophagy induced by rhArg in a time-dependent manner. Moreover, rhArg could induce ERK1/2 activation in a dose- and time-dependent manner and rhArg-induced autophagy was attenuated when p-ERK1/2 was inhibited by MEK 1/2 inhibitor, U0126. Taken together, this study provides new insight into the molecular mechanism of autophagy involved in rhArg-induced cell apoptosis and growth inhibition, which facilitates the development of rhArg in combination with CQ as a potential therapy for malignant melanoma.     

High dose of ascorbic acid induces cell death in mesothelioma cells

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.     

Anticarcinogenic effects of halofuginone on lung‐derived cancer cells

Malignant mesothelioma is a rare but aggressive form of malignancy, which is difficult to diagnose and is resistant to current chemotherapeutic treatment options. Molecular techniques have been used to investigate the mechanisms of action and the beneficial therapeutic effects of halofuginone (HF) in several cancers but not malignant mesotheliomas. In this study, the antiproliferative and apoptotic effects of HF were investigated through its ability to deregulate EGFR downstream signalling cascade proteins in the pathologically aggressive malignant mesothelioma and non‐small‐cell lung cancer cells. We showed that administration of HF at nanomolar concentrations induced a dose‐dependent reduction in the viability of cancer cells, made cell cycle arrest, inhibited proliferation of cancer cells via STAT3 and ERK1/2 pathways and triggered the apoptotic cascade via p38MAPK. We demonstrated that the apoptotic cell death mechanism was mediated by enhanced activation of caspase‐3 and concomitant PARP cleavage, downregulation of Bcl‐2 and upregulation of Bax in both malignant mesothelioma and lung cancer cells. In particular, we demonstrated that cancer cells were more sensitive to HF treatment than normal mesothelial cells. Taken together, this study suggests that HF exerts its anticancer effects in lung‐derived cancers by targeting signal transduction pathways mainly through deregulation of ERK1/2, STAT3 and p38MAPK to reduce cancer cell viability, induce cell cycle arrest and apoptotic cell death. Thus, HF might be considered as a potential agent against malignant mesothelioma and/or lung cancer cells.     

ATRA inhibits the proliferation of DU145 prostate cancer cells through reducing the methylation level of HOXB13 gene

All-trans retinoic acid (ATRA) has been widely investigated for treatments of many cancers including prostate cancer. HOXB13, silenced in androgen receptor-negative (AR(-)) prostate cancer cells, plays a role in AR(-) prostate cancer cell growth arrest. In this study we intended to elucidate the mechanisms that are involved in the proliferation inhibition of AR(-) prostate cancer cells triggered by ATRA. We discovered that ATRA was able to induce the growth arrest and to increase HOXB13 expression in AR(-) prostate cancer cells. Both EZH2 and DNMT3b participated in the repression of HOXB13 expression through an epigenetic mechanism involving DNA and histone methylation modifications. Specifically, EZH2 recruited DNMT3b to HOXB13 promoter to form a repression complex. Moreover, ATRA could upregulate HOXB13 through decreasing EZH2 and DNMT3b expressions and reducing their interactions with the HOXB13 promoter. Concurrently, the methylation level of the HOXB13 promoter was reduced upon the treatment of ATRA. Results from this study implicated a novel effect of ATRA in inhibition of the growth of AR(-) resistant human prostate cancer cells through alteration of HOXB13 expression as a result of epigenetic modifications.     

ZEBULARINE: A UNIQUE MOLECULE FOR AN EPIGENETICALLY BASED STRATEGY IN CANCER CHEMOTHERAPY. THE MAGIC OF ITS CHEMISTRY AND BIOLOGY

1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.     

Anticancer drugs induce hypomethylation of the acetylcholinesterase promoter via a phosphorylated-p38-DNMT1-AChE pathway in apoptotic hepatocellular carcinoma cells

Apoptosis, also known as programmed cell death, plays an essential role in eliminating excessive, damaged or harmful cells. Previous work has demonstrated that anticancer drugs induce cell apoptosis by inducing cytotoxicity. In recent years, several reports demonstrated modulated expression of DNA methyltransferases 1 (DNMT1) and acetylcholinesterase (AChE) in a variety of tumors. In this study, we showed that the expression of DNMT1 was decreased and the methylation of CpGs in the promoter of AChE was reduced in anticancer drugs-induced apoptotic hepatocellular carcinoma cells. Silencing of DNMT1 expression by AZA or RNA interference (RNAi) restored AChE production and inhibition of AChE expression by RNAi protected HCC cells from anticancer drugs-induced apoptosis. Furthermore, we demonstrated that the regulation of AChE by DNMT1 was involved in the phosphorylated p38 pathway in anticancer drugs-induced apoptosis. In addition, immunohistochemical staining showed that P-p38, DNMT1 and AChE were aberrantly expressed in a subset of HCC tumors. Taken together, we demonstrated the regulation of AChE by DNMT1 and further, we found that this regulation was involved in the phosphorylated p38 pathway in anticancer drugs-induced apoptosis.     

Incorporation of Zebularine from its 2′-Deoxyribonucleoside Triphosphate Derivative and Activity as a Template-Coding Nucleobase

Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) was studied as both a 2 ′-deoxyribosyl 5 ′-triphosphate derivative and as a template incorporated into an oligonucleotide. Using a novel pyrosequencing assay, zebularine acted as cytosine analog and was incorporated into DNA with a template pairing profile most similar to cytosine, pairing with greatest efficiency opposite guanine in the template strand. Guanine was incorporated with greater affinity than adenine opposite a zebularine in the template strand. Computer modeling of base-pairing structures supported a better fit of zebularine opposite guanine than adenine. Zebularine acts as a cytosine analog, which supports its activity as an inhibitor of cytosine methyltransferase.     

Mutagenicity of the cytidine analog zebularine in Escherichia coli

We have examined the mutagenic properties of zebularine, a cytidine analog lacking the amino group at C-4 that has potential use in chemotherapy. Because the hydrate is a strong inhibitor of cytidine deaminase, its use can enhance the potency of other cytosine based compounds such as 5-azacytidine (5AzaC) and cytosine arabinoside (ara-C) that are inactivated by cytidine deaminase. Using the newly developed rpoB/Rifr system in Escherichia coli, we examined base substitution mutations caused by zebularine in the chromosomal rpoB gene. Zebularine is a potent mutagen that causes mainly G : C --> A : T transitions and favors certain hotspots. Mutations are not specific to the rpoB gene, since there is also a strong induction of mutations in the thyA gene. In the absence of mismatch repair, zebularine induces both base substitutions and frame shifts at rates well above those seen in wild-type strains treated with zebularine or in mismatch repair deficient strains without treatment. The nature of these induced mutations indicates that zebularine is stimulating the induction of increased replication errors, in addition to the targeted G : C --> A : T mutations, and that these errors are normally repaired by the mismatch repair system.     

Mechanistic Insights on the Inhibition of C5 DNA Methyltransferases by Zebularine

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)—the enzymes responsible for DNA methylation—are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(β-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.     

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. The magic of its chemistry and biology

1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.