It is now widely accepted that cancer is both a genetic and epigenetic disease. Along with genetics, epigenetics function as an additional explanation that the complexity of abnormal changes arised in cancer cells. Epigenetics describe a hereditable alteration in gene expression independent of a change in the DNA sequence.1 DNA methylation is essentially mediated by DNA methyl transferases (DNMT) with S-adenosyl-methionine (SAM) as the methyl donor.2 Aberrant DNA methylation in cancer cells is characterized by focal CpG island hypermethylation and diffuse genomic hypomethylation. In cancer cells, there was a 20%-60% of less methylated CpG island than in normal cells. This loss of methylation, which increases through the evolution of a tumour from a benign lesion to an invasive malignancy, is mainly resulted from the demethylation of repetitive DNA sequences, coding regions and introns of genes.2,3 However, it is to be determined how important DNA hypomethylation is in carcinogenesis. Probably it is a truly causative factor in some cancers and only a modulator of tumour risk in others. In contrast to the uncertainties of the roles of hypomethylation in tumour formation and progression, DNA hypermethylation is an epigenetic modification that can play an important role in the control of gene expression in mammalian cells, and its role in carcinogenesis has been a topic with considerable interest in recent years.1,4,5 In many types of tumours, hypermethylation of CpG islands in the promoter region of different tumour suppressor genes has been observed and correlates closely with loss of mRNA or protein. To our best knowledge, the panel of genes suffering from methylation and silencing in different cancers is always increasing. In fact, all important cellular signaling pathways in relation to carcinogenesis can be affected by methylation of specific genes.2 Thus, reactivation of tumour suppressor genes by DNA methylation inhibitors has become a potential and promising area of cancer therapy.1,6,7
DNA METHYLATION INHIBITORS
Promoter hypermethylation can be targeted by inhibitors of DNMT, which can be divided into nucleoside analogues and non-nucleoside analogues. The first, most widely used and more effective DNMT inhibitors are represented by nucleoside analogues of cytidine in which the cytosine ring has been modified to endow them with DNMT inhibitory activity; they include 5-azacytidine, 5-aza-2′-deoxycytidine (5-aza-CdR), 5,6-dihydro-5-azacytidine and zebularine. Nucleoside deoxycytidine analogues once known as classic cytotoxic agents and recently gained lots of attention as a demethylating agent for the treatment of hematological malignancies.8 Use of azanucleosides, such as 5-azacytidine and 5-aza-CdR, in cell culture produces re-expression of certain genes, which otherwise were repressed in association with hypermethylated CpG-rich promoters. Hence the notion developed that 5-azacytidine and 5-aza-CdR is a demethylating agent. However, this kind of agents exerts poor activity on solid tumors, for example, gastrointestinal malignancies.9 A recent study disclosed that (1) 5-aza-CdR has nothing to do with DNA demethylation; (2) it can not prevent even de novo methylation in non-replicating cells; (3) it can only prevent replication coupled maintenance as well as de novo methylations. Finally, the authors suggestted that terming/designating 5-aza-CdR as DNA demethylating agent is a serious misuse of chemistry and chemical terminology.10 In addition, the common adverse effect such as myelosuppression and severe gastrointestinal events and the instability in aqueous solutions also limits their application. Zebularine is much less cytotoxic than other nucleoside analogues, however, it requires prolonged exposure to high doses of the compounds, the rate and extent of the in vivo metabolism is fairly low. With these acknowledged limitations, It seems that Zebularine could not be a better alternative than its counterparts.11
Non-nucleoside inhibitors of DNMT function without being incorporated into the DNA. Hence they are theoretically less toxic than the nucleoside analogues. They include: procaine, mitoxantrone, N-acetyl-procainamide, procainamide, hydralazine, and the main polyphenol compound of the green tea (-) -epigallocatechin-3-gallate (EGCG). It has been suggested that procaine inhibits DNMT by perturbing interactions between the protein and its target sites.12 Hydralazine and procainamide were first reported to have DNA methylation-inhibition effect in 1988.13 A recent study indicated that hydralazine can inhibit cell growth in human cervical cancer in vitro by inhibition of APC promoter methylation and reactivation of APC expression, and can be potentially used for the clinical treatment of human cervical cancer.14 It has been proposed that the so-called “non-toxic and orally administered agent” EGCG could also block the catalytic pocket of the human DNMT.15 It was newly demonstrated that EGCG has selective anti-angiogenic effects on tumor-associated endothelial cells and endothelial progenitor cells.16 By encapsulating EGCG in polylactic acid-polyethylene glycol nanoparticles, EGCG retains its biological effectiveness with over 10-fold dose advantage for exerting its proapoptotic and angiogenesis inhibitory effects.17
Despite the promising results in preclinical trials, the applicability of non-nucleoside DNA methylation inhibitors to humans has met with limited success. A recent study compared the activity of different nucleoside with non-nucleoside inhibitors of DNMT and found a functional diversity among them.18 Nucleoside analogues, particularly 5-aza-CdR, displayed the most significant demethylating efficiency and were the only able to cause DNA promoter region demethylation and tissue inhibitors of metalloproteinase (TIMP)-3 re-expression. Also in a systemic study comparing the biological effects of non-nucleoside inhibitors versus 5-aza-CdR, the authors examined EGCG, hydralazine and procainamide, and compared their effects and potencies with 5-aza-CdR. They found that 5-aza-CdR is far more effective in DNA methylation inhibition as well as in reactivating genes, compared with non-nucleoside inhibitors.19
In addition to nucleoside and non-nucleoside analogues, there has been increasing interest in the development of small molecules targeting DNMT. Theoretically, most small-molecule chemotherapeutic agents have more advantages in that the DNA incorporation requirement of nucleoside analogues makes these agents exquisitely S-phase specific, a considerable barrier in some neoplasms. However, the DNMT family have so redundant functions that inhibiting more than one member is required to optimally activate tumour-suppressor genes. A recent study suggested that MG98, an antisense compound to DNMT1, could partially down-regulate DNMT1, but no objective clinical response was observed.20 In both phase I and phase II trials in renal cancer, MG98 has shown a lack of efficacy and non-linear pharmacokinetics which in turn resulting in transaminitis and lack of correlation between levels of methylation and toxicity.21 RG108 is another small-molecule inhibitor of DNA methylation which showing DNMT inhibitory activity. Studies suggested that RG108 may be less toxic and more specific for hypermethylated tumour suppressor genes.22,23
ISSUES TO BE CONSIDERED INVOLVING DEMETHYLATING AGENTS
When designing clinical trials involving demethylating agents, it is crucial to recognize that although DNA methylation inhibitor is a molecularly precise targeted therapy approach, the downstream effects on neoplastic behaviour are quite diversified, sometimes even conflicting. These agents could have different effects depending on the pattern of genes methylated in a given tumour and argues that patients stratification according to their methylation status may be necessary in clinical trials of demethylating agents. First, hypermethylation at DNA promoter region does not necessarily lead to gene silencing. In some situations, DNA methylation may not be involved in epigenetic gene silencing as trimethylated H3K27 (H3K27trim) which has been shown to silence tumour suppressor genes independent of promoter methylation.24 Like many human genes, the telomerase reverse transcriptase (hTERT) gene, which is involved in cellular immortalization and transformation, contains a high density of CpG islands in its promoter region. Surprisingly, some studies suggest that increased DNA methylation in this area in hTERT-positive cancer cells and lack of methylation in normal hTERT-negative cells.25 Second, some recent studies now point to promoter methylation being a secondary event triggered by decreased gene expression.26 Third, promoter hypermethylation of different tumour suppressor genes may correlates with diversified clinical prognosis. Methylation of the DNA mismatch repair gene human mutL homologue 1 (hMLH1) occurs in cisplatin-resistant ovarian cell line models.27,28 Apoptotic protease activating factor 1 (apaf1) represents another gene whose methylation may increase resistance to chemotherapy29,30 Methylation of apaf1 in melanoma cells can be reversed by 5-aza-CdR, leading to increased apaf1 transcription and increased the chemosensitivity.30 In contrast, methylation of the fancf gene was observed in ovarian cancer cell lines with a defective BRCA2 pathway and increased sensitivity to cisplatin. Treatment with 5-aza-CdR led to demethylation of the fancf gene and reduced sensitivity towards cisplatin in these cell line models.31 A clinical trial suggests methylation of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (mgmt) in the promoter region is an independent predictor of longer survival for glioblastoma patients treated with a methylating agent (temozolomide) as well as radiation.32 Promoter hypermethylation of mgmt also correlated with increased survival of patients with diffuse large B-cell lymphoma after chemotherapy that included cyclophosphamide.33
Another major concern regarding the application of DNA methylation inhibitors in the clinic is whether they also affect the epigenetic silencing patterns of normal cells, and whether they would activate the transcription of oncogenes. Like many other therapeutics currently being developed against specific diaeases, DNA methylation inhibitors are hoped to work in some specific way, and thus have less side/adverse effects than the nonspecific conventional chemotherapy. Despite the promising results of some clinical studies, these agents can seem paradoxical in anticancer therapies because many tumours are characterized by a global DNA hypomethylation.3 We have no idea what effect prolonged demethylation and epigenetic activation has on other non-tumour related genes, especially in the normal tissues. Some investigators have raised the concern that the longer term use of demethylating agents could in itself be carcinogenic.33 An in vitro study found that 5-aza-CdR stimulated the invasion potential of pancreatic cancer through reactivation of invasion- promoting genes.35 Another study suggested that 5-aza-CdR slowed the growth of tumours in nude mice, however, it also induced a battery of prometastatic genes, namely, uPA, cxcr4, heparanase, synuclein γ, and transforming growth factor-beta (TGF-β), by demethylation of promoters.36
The combined anticancer therapy may ultimately offer the most successful approaches. The combined strategy may contribute to reduction in the dose of each drug and reduce the side/adverse effects of each individual agent. As suggested in a most recent study, DNA demethylating interventions alone are not able to restore a complete euchromatic status and a full transcriptional reactivation of the epigenetically silenced tumour suppressor genes, and emphasize the necessity of targeting multiple elements of the epigenetics machinery for a successful treatment of malignancies.37 Further study found that combined treatment with the 5-aza-CdR and the histone deacetlyase (HDAC) inhibitor valproic acid (VPA) efficiently prevented medulloblastoma and rhabdomyosarcoma formation, whereas monotherapies with either drug were less effective. However, the treatment was not effective in clinically overt, advanced stage tumours.38
There is no doubt that combination of traditional cancer chemo- and/or radio-therapy with the use of epigenetic therapy, restore the status of DNA methylation and histone acetylation, will occupy a huge potential for successful cure of malignancies in future. Such approaches might also help to sensitize cancer cells, especially cancer stem cells, which are refractory to standard chemotherapy. However, there must be still a long way to walk before DNA methylation inhibitors can function well like specific targeted anticancer drugs as we expected. Hence far more further studies are needed. It is important to get a better understanding of the diverse molecular mechanisms of the demethylating agents available today. If the exact methylation profiles of tumours are clarified and agents targeting the specific genes are available, then the treatment of cancer could be more effective and rational.
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