Methylation inhibitors for cancer prevention -
The development of cancer is a complex, multifactorial process characterized mainly by genetic mutations and epigenetic alterations. Cancer is a disease that can take many years to develop, from initiation to progression.
For example, all the common epithelial cancers lung, colorectal, breast, prostate, pancreas and ovary have a long latency period, often 20 years or more. By the time they are clinically detectable, the cells may harbor hundreds of mutations in different genes Sporn The long-term development of certain types of cancer could represent a major opportunity to use multi-functional, multi-targeted preventive drugs in order to block or reverse cancer-related modified cells.
One path for cancer prevention could be to target and reverse the early epigenetic alterations that, unlike genetic mutations, are potentially reversible and can be restored to their normal state.
The epigenetic mechanisms are regulating gene expressions through genomic DNA methylation, histone post translational modifications, chromatin remodeling, and expression of non-coding RNAs microRNAs and long non-coding RNAs.
Each epigenetic mechanism is controlled by specific protein classes which attach, remove, or maintain specific chemical groups that constitute epigenetic marks for activation or inactivation of the gene transcription.
When this regulatory circuit is discontinued by internal or external factors, normal physiological functions are affected, leading to tumor initiation process Timp and Feinberg Recent advances made in epigenetic field and cancer research showed that genetic and epigenetic mechanisms are not separate events in cancer; they interconnect and influence each other during tumorigenesis You and Jones Alterations in epigenetic mechanisms can lead to genetic mutations; genetic mutations in epigenetic regulators lead to an altered epigenome Timp and Feinberg Furthermore, evidence have suggested that epigenetic modifications might occur early in tumorigenesis and some of them even precede genetic mutations during cancer initiation Feinberg et al.
The abnormal proliferation of cells, due to accumulation of genetic and epigenetic aberrations, causes deregulation of major cellular processes, including cell cycling, DNA damage response, differentiation, and apoptosis. Epidemiological studies Zamora-Ros et al.
Over the recent years, studying the effects of bioactive nutrient treatment on the epigenome has become widespread, and it is currently certain that they can modulate epigenetic mechanisms of gene expression, such as genomic DNA methylation, acetylation or methylation of lysine residues from histones H3 and H4.
Changes in DNA methylation have been recognized to be among the most common molecular alterations in human neoplasia and hypermethylation of gene-promoter regions is being revealed as one of the most frequent mechanisms of gene function loss. DNA methylation is the major epigenetic mechanism that provides a stable and reversible mechanism for gene silencing; it plays an important role in regulating gene expression, chromatin architecture and chromosome stability.
DNA methylation at the 5 position of cytosine 5-mC is a key epigenetic mark that is critical for various biological and pathological processes.
It consists in the addition of a methyl group from the universal methyl donor, S-Adenosyl-methionine SAM , to the cytosine at the CpG dinucleotide residues. The members of the DNA methyltransferase DNMT family directly catalyze the addition of methyl groups onto DNA and are the major players as epigenetic modifiers.
DNMT3A and DNMT3B are de novo methyltransferases by initiating DNA methylation and establishing the methylation patterns independently of replication. DNMT1 maintains the original pattern of DNA methylation in a cell lineage manner, and has the ability to repair DNA methylation Mortusewicz et al.
The maintenance mechanism mediated by DNMT1 is crucial to ensure the faithful reestablishment of 5-mC on the newly synthesized strand after DNA replication Mortusewicz et al. Moreover, there is an active cooperation between all three enzymes in order to maintain DNA methylation at densely methylated regions, repetitive elements, and imprinted genes Liang et al.
Unmethylated CpG islands correspond to either active transcription or a poised state, where genes can be expressed if the appropriate molecular signals are present Suzuki and Bird These methylation patterns of the genome are vital for both chromosomal and genomic stability, possibly through the repression of retroviral transposons Jones Consequently, the epigenetic mechanism through DNA methylation facilitates the organization of the genome into active euchromatin and inactive regions heterochromatin with respect to gene transcription.
DNA methylation is essential for normal mammalian development, function and differentiation by its epigenetic control of protein-coding RNAs or non-coding RNAs expression. The 5-mC epigenetic mark is chemically stable and its presence at the promoter sites induces transcriptional inhibition by sterically blocking the further binding of transcription factors Deaton and Bird Sequentially, the MBD proteins are recruited at methylated DNA and further facilitate the formation of compact, inactive heterochromatin.
The sequential recruitment of different protein complexes, followed by histone acetylation, demonstrated that the formation of open transcriptionally active chromatin is a dynamic process where layers of epigenetic regulators participate to the gene expression mechanism Memedula and Belmont ; Bintu et al.
For decades, DNA methylation has been considered to be a non-reversible reaction, until the discovery of 5 hydroxymethylcytosine 5-hmC and the TET family of enzymes. The active demethylation consists of a series of successive oxidation reactions catalyzed by TETs. First, 5-mC is converted to 5-hmC; afterwards, the methyl group is removed by a TDG-mediated base excision mechanism Oswald et al.
The 5-hmC epigenetic mark is also associated with differentiation and normal development, Ficz et al. While 5-mC is associated with closed heterochromatin, 5-hmC is associated with DNA demethylation and an open active chromatin state. The role of 5-hmC in gene expression regulation has still not been fully elucidated.
The unique genomic distribution patterns of TET1 and 5-hmC at the transcription starting sites and promoters, as well as gene bodies, are suggesting that they might regulate gene expression through modulating chromatin accessibility or by inhibiting repressor binding Williams et al.
As an epigenetic reader protein, methyl-CpG binding protein 2 MeCP2 has similar affinity to both DNA epigenetic marks Mellén et al. One explanation could be that the binding of MeCP2 to 5-mC can possibly hinder the production of 5-hmC Mellén et al.
Therefore, 5-hmC and MeCP2 might constitute a cell-specific epigenetic mechanism for the regulation of gene expression and remodeling the chromatin structure.
The interconnection between DNA methylation and the demethylation processes is exemplified in Fig. DNA methylation and demethylation process. DNMTs catalyze the transfer of the methyl group to cytosine and generate 5-mC using SAM as methyl donor and producing SAH. In the first step of demethylation process, the 5-mC is converted to 5-hmC, and after several oxidation reactions the methyl group can be remove by TDG-mediated base excision repair mechanism.
DNA methylation is a reversible, enzymatically controlled mechanism of gene expression, involved, among other processes, in normal embryogenesis, tissue differentiation and chromosome stability.
Modifications in any of the layers controlling this epigenetic process can lead to carcinogenesis, as discussed in the following section. Cancer methylation has been characterized by global hypomethylation together with local, de novo promoter CpG islands hypermethylation Jones and Baylin ; Sharma et al.
Furthermore, some local variations in methylation pattern at only several key genome loci are sufficient for cancer initiation Plass et al. Importantly, the altered patterns of DNA epigenetic marks 5-mC, 5-hmC are frequently accompanied by a critical imbalance in transcriptional programs involving differentiation and stem cell maintenance, thereby could participate to tumor initiation and sustaining cancer cells growth Jones and Baylin Actually, a series of studies showed that the DNA methylation is a driver of tumorigenesis and that cancer cells suffer additional epigenetic alterations which are essential for cancer cell survival Baylin and Herman ; De Carvalho et al.
Furthermore, the fact that DNA hypermethylation could be an early event in carcinogenesis is supported by the finding that adjacent normal tissues also harbor altered DNA methylation patterns Taby and Issa For example, the hypermethylation and silencing of the intracellular detoxification enzyme GSTP1 is considered a molecular hallmark of prostate cancer and was already implemented in clinical diagnosis.
The loss of GSTP1 enzymatic detoxification activity may explain the well-known sensitivity of human prostatic carcinogenesis to environmental factors and the demethylation of GSTP1 promoter could become a target for epigenetic chemoprevention Jerónimo et al.
Similarly, methylation mediated silencing was reported in other important DNA repair genes, such as MGMT, BRCA1, BRCA2 and MSH2 at a pan-cancer level, and are frequently observed in multiple cancer types, for instance lung, gastric, colorectal, leukemia, brain, liver, breast, and prostate Witte et al.
The inactivation of genes in DNA repair pathways will further propagate the carcinogenic state by allowing cells to accumulate additional genetic lesions.
MGMT, which normally protects from mutations occurring at guanine bases, is silenced by hypermethylation events and often occurs early in tumorigenesis Witte et al. These results suggest that a primary epigenetic defect in mismatch repair mechanisms can accelerate the rate of accumulation for additional mutations in cancer cells.
In addition, the silencing of transcription factors could indirectly silence or downregulate a large number of other genes. For example, silencing a key tumor suppressor p16 encoded by the CDKN2A gene in humans is related with aberrant promoter hypermethylation and it is a common epigenetic mark in human cancers.
Mice bearing a hypermethylated p16 promoter had a higher incidence of developing spontaneous cancer during ageing. But when the mice carried inactivating germline mutations in one allele of p16 and epigenetic alterations in the other allele, they showed early onset of tumors and shorter survival time Yu et al.
Put together, these data demonstrate that epigenetic mutations are able to act as driver events in tumor initiation and progression. Interestingly, dysregulation of DNA methylation status at promoters of non-coding RNAs miRNAs and lncRNAs could also promote carcinogenesis Kozaki and Inazawa ; Pop et al.
Aberrant microRNA expression in cancer has been associated with epigenetic regulation, such as DNA methylation and histone modifications. Also, microRNAs control and regulate expression of major epigenetic modifier proteins involved in DNA methylation processes, including DNMTs and TETs. Several studies have demonstrated that alterations in the expression of miRNAs are prominent events during the early stages of liver carcinogenesis and may predict susceptibility to cancer development Anwar and Lehmann In vivo studies showed that a methyl-deficient diet induced hepatocellular carcinogenesis associated with global DNA hypomethylation, and with changes in several miRNA expression, which could be reversed by restoring dietary methyl donors Parasramka et al.
The global lower methylation level hypomethylation is a common epigenetic alteration in cancer, especially at Long Interspersed Nuclear Element LINEs regions, which are a group of retrotransposons widespread in human genome.
They are translated into proteins that act as reverse transcriptase able to reproduce DNA copies, which are then relocated into new genomic sites. In human somatic cells, the LINE-1 is heavily methylated and thus is mostly suppressed, maintaining the genomic stability by avoiding retrotransposition to other genomic loci Lee et al.
Several studies showed that LINE-1 is gradually hypomethylated during cancer progression, with the first signs of global methylation changes initiated early in carcinogenesis Slotkin and Martienssen ; Kitkumthorn and Mutirangura Hypomethylation of LINE-1 triggers the active process of genome reorganization, and the relocations of these interspersed repetitive sequences are a source of endogenous mutagenesis and polymorphism in the premalignant and malignant cells Kitkumthorn and Mutirangura ; Lee et al.
Also, the loss of 5-hmC is an epigenetic hallmark of aggressive tumors, such as melanoma, glioblastoma or ovarian cancer, with both diagnostic and prognostic implications Tucker et al.
The overexpression of active proteins IDH2 or TET2 in animal models for human melanoma was an efficient way to increase the 5-hmC level Lian et al.
Likewise, pre-treatment with DNMTs inhibitors restored the 5-hmC patterns via enhanced levels of TET family enzymes, both in vitro and in vivo experiments Tucker et al. There are approximately 40 epigenetic regulators that exhibit some form of alteration in cancer Jones and Baylin The most prominent are somatic mutations in the proteins involved in DNA methylation and demethylation mechanisms.
The epigenetic regulators expression could be directly altered in various cancers, but other mutated genes may impinge on the proper function of these enzymes.
For example, recurrent mutations in the IDH1 and IDH2 alter their enzymatic activity and consequently the 2-hydroxyglutarate is produced, which may inhibit several dioxygenases, including TET2 and KDM4C Feinberg et al.
This indicates that not only the epigenetic factors are directly altered in tumorigenesis, but also that mutations in other pathways can have an impact on the regulation of gene expression. Genetic alterations in TET1 and TET2 have also been identified in leukemia and solid tumors Wu and Zhang , with direct effects on epigenetic degradation of 5-hmC and 5-mC patterns.
Coordinated epigenome changes can also be achieved via the interaction of multiple epigenetic regulators, which guide different enzymatic activities to the same locus.
Recent results have demonstrated that the PRC2 complexes are recruited to specific DNA regions based on DNA sequence and transcription factor occupancy.
The protein EZH2, that mediates repressive chromatin formation through the deposition of H3K27 methylation, and DNMT enzymes, act at the same locus for the coordination of repressive histone and DNA methylation marks Viré et al.
Thus, the crosstalk between the different layers of the epigenetic mechanism could amplify early epigenetic changes, leading to the development of cancer. The overexpression of DNMTs represents a common feature in a variety of tumors, and results in local DNA hypermethylation and oncogenic activation.
Highly expressed DNMT3A and DNMT3B have been found in a large number of patient specimens, with increased DNMT3A expression in hepatocellular carcinogenesis, where intense hypomethylated genomic regions were also observed Zhao et al.
Moreover, high expression levels of DNMT3B have been correlated with the epigenetic inactivation of BRCA1 in sporadic breast tumors Butcher and Rodenhiser As a result, the overexpression of DNMTs in many cancers promoted local and global DNA methylation aberrations related with genomic instability and oncogenic pathways activation.
In conclusion, there are several epigenetic mechanisms related to DNA methylation patterns, from local hypermethylation of specific gene promoters to global DNA hypomethylation, with impact on cellular processes which dysregulated can lead to carcinogenesis, as we exemplified in Fig.
Epigenetic alterations of DNA methylation in carcinogenesis. There are two main epigenetic changes of DNA methylation related to cancer initiation and development—global DNA hypomethylation and local gene promoters hypermethylation.
Each leads to specific events, such as activation of LINEs or, inactivation of genes involved in specific cellular processes: detoxification, DNA repair genes, and tumor suppressor gene respectively.
Isolated or in summation, these alterations can eventually lead to cancer. Whether dysregulation of DNMTs and TETs enzymes can be modulated by phytochemicals in an efficient manner, in order to counteract various mechanisms acting in tumor initiation and progression, will be the topic of the following section.
Cancer chemoprevention implies the use of dietary or pharmacological compounds to prevent, inhibit, or even reverse the process of carcinogenesis before clinical manifestation of the disease. Therefore, effective chemoprevention requires the use of compounds that inhibit specific molecular steps in the carcinogenic pathway, including the epigenetic alterations that are early and potentially reversible events.
Substantial experimental evidence and epidemiological studies indicate the potential importance of dietary phytochemicals and nutritional factors in cancer prevention.
Natural products with bioactive components have gained increasing attention in cancer prevention and therapy, due to their compatibility with biological target sites and less induced toxicity to normal cells Remely et al.
Several preclinical studies have reported that many phytochemicals with anti-inflammatory, anti-oxidation and anti-proliferative properties can prevent cancer initiation and development by inducing apoptosis and activating antioxidant enzymes Venkatachalam et al.
However, the translation of chemopreventive properties of phytochemicals to clinical practice has not been yet achieved. Among the impressive number of phytochemicals with anti-tumoral properties, some polyphenols and organosulfur compounds are part of the dynamic interaction between the genome and the environment with specificity at physiological concentrations, and well known to modulate mechanisms underlying in human health.
Recent studies have highlighted the cross-talk between cancer metabolism and the epigenome. Metabolites such as SAM, acetyl-coA, and AMP are required for epigenetic mechanisms such as DNA and histones methylation, histone acetylation or phosphorylation Donohoe and Bultman ; Newman and Maddocks The metabolic pathways and enzymes that supply these key compounds are therefore critical for the maintenance and adaptation of the epigenome.
Indeed, a diet with deficit in methionine decreases SAM levels, leading to diminished DNA and histone methylation with significant effects upon gene expression Donohoe and Bultman ; Parasramka et al.
The metabolism of folate, betaine, choline, and methionine are interrelated, and the deficiency of one nutrient can cause metabolic and functional disturbances.
A diet poor in methyl donor contributors can have a rapid effect on global DNA methylation pattern. For example, within 1 week, the global DNA hypomethylation and increased levels of mRNA for oncogenes c-fos and c-myc were observed in liver tissues of Fischer rats fed with methionine and choline-deficient diet.
After restoration of proper methyl donors rich diet, the global and local DNA methylation pattern returned to normal within 1—2 weeks Niculescu and Zeisel In the case of longer exposure to methyl deficient nutrition 18—36 weeks , the epigenetic alterations of DNA methylation could not be reversed by reintroducing the animal models to the right diet.
The global DNA hypomethylation pattern and altered hepatic foci in their liver were irreversible Pogribny et al. These data provide further experimental evidence to demonstrate that epigenetic alterations may contribute to the initiation and promotion of liver carcinogenesis. During methionine starvation, the other metabolic pathways, such as serine cycle could provide cofactors to recycle homocysteine to methionine.
Indeed, the serine-dependent de novo ATP synthesis might support the conversion of methionine to SAM. In this case the ATP pool is reduced and this can have a direct impact on the rate of SAM generation and methylation of DNA Maddocks et al.
Recently, the specific contribution of one-carbon metabolism-dependent DNA methylation in pancreatic cancer has been explored. The loss of the serine—threonine kinase, LKB1, promotes tumorigenesis in KRAS mutant pancreatic cancer, accompanied by increased levels of global DNA methylation and increased expression of DNA methyltransferases for which SAM is a critical cofactor.
This serine-dependent DNA methylation upon the loss of LKB1 in KRAS-mutant cells contributes to tumor growth, presumably through the activation of several oncogenes Kottakis et al. Dietary and genetic perturbation of metabolic pathways could lead to dysfunctional DNA synthesis and DNA methylation, connecting directly the cancer metabolism to the epigenetic mechanism.
The cellular DNA methylation processes involve a series of catalytic reactions, which result in the generation of the principal methyl donor, SAM, followed by methyl group transfer reactions. As a consequence of methyl group transfer, SAM is converted to SAH, which binds to methyltransferases and induces product inhibition Mortusewicz et al.
Therefore, maintaining the proper ratio of SAM to SAH is a determinant factor for DNA methylation mechanism, since this ratio dictates methyltransferase activity in vivo. Disturbance of this system may be caused by dietary imbalances and in consequence the major epigenetic regulatory enzymes are affected, dysregulating DNA methylation pattern Stefanska et al.
In a pre-malignancy pathological condition, the appropriate consumption of a diet rich in methyl donor nutrients may interfere with early carcinogenesis events leading to cancer prevention.
The flavanol-rich diets contain polyphenols with catechol structures that can be methylated by catechol- O -methyltransferase COMT enzyme using SAM as a methyl donor Bistulfi et al. This methylation reaction results in the demethylation of SAM and formation of SAH, which is a potent and selective inhibitor of DNA methyltransferase Zhu et al.
Phytochemicals and other bioactive dietary compounds can restore global and gene-specific promoter DNA methylation patterns by reactivating DNA methyltransferases or providing the provision of methyl groups. Therefore, phytochemicals could epigenetically modulate gene expression by changing the chromosomal integrity and stability with benefits on health conditions.
Numerous studies have demonstrated that certain dietary phytochemicals inhibit tumor growth by affecting epigenetic signaling pathways both in vitro and in vivo Remely et al. The dietary phytochemicals with epigenetic modulation activities of DNA methylation levels can be categorized in three group based on their mechanism of action Ho et al.
Polyphenols are the largest class of plant secondary metabolites that are mainly found in fruits, vegetables, cereals, and beverages. Besides common antioxidant or anti-inflammatory activity, many polyphenols might modulate early epigenetic alterations related to cancer prevention. The polyphenol groups include phenolic acids hydroxybenzoic and hydroxycinnamic acids , lignans, stilbenes, and flavonoids Hardman Flavonoids are the most representative group of dietary polyphenols with diverse biological activities including anti-bacterial, anti-viral, analgesic, hepatoprotective, apoptotic, and estrogenic functions Kumar and Pandey The chemoprevention activity of flavonoids might be mediated by certain epigenetic mechanisms, including modulation of DNA methylation status and histone methylation and acetylation Jiang et al.
The flavone apigenin can restore the silenced status of Nrf2 gene in skin epidermal cells by reducing the expression of three DNA methyl transferase proteins DNMT1, DNMT3A, and DNMT3B as well as the expression of some HDACs Shukla and Gupta In addition, apigenin, together with another flavone, luteolin, has been reported to act synergistically to modulate the DNMT activity in esophageal squamous cell carcinoma line Busch et al.
Research suggests that a diet rich in flavones might decrease the risk of certain cancers, including breast, digestive tract, skin, and prostate cancer Li and Tollefsbol ; Shukla and Gupta Genistein is the most potent DNMT inhibitor amongst isoflavones, capable to reactivate methylation silenced genes such as RARb, p16INK4a, and MGMT in esophageal squamous carcinoma and prostate cancer cells Fang et al.
The major catechin from green tea, EGCG, exerts its chemoprevention effect by blocking cell proliferation and transformation and promoting apoptosis and cell cycle arrest in several human cancer cell lines including leukemia, melanoma, breast cancer, lung, and colon Singh et al. Additionally, EGCG has been demonstrated to induce the increase of tumor suppressor expression, such as: p53, p21, p16 and Rb with certain roles in chemoprevention Pandey et al.
Moreover, EGCG possesses a chemopreventive effect against a broad spectrum of carcinogens by inhibiting the chemical induced colon, liver and skin carcinogenesis in several animal models Henning et al. EGCG exerts its epigenetic modulator capacity of DNA methylation processes indirectly, by acting as a substrate for COMT catalyzed methylation reaction Bistulfi et al.
Molecular docking studies indicate that the gallic acid moiety of EGCG can accommodate in the hydrophilic active pocket of DNMT1 Lee et al. Fang et al. demonstrated that EGCG binds to DNMT and competitively inhibits the enzymatic activity yielding to the reactivation of methylation-silenced genes in prostate cancer cells Fang et al.
In addition, treatments of different PCa cell lines with EGCG have determined a dose- and time-dependent re-expression of GSTP1 enzyme concomitantly with the down-regulation of DNMT1 Naponelli et al. Recent studies have demonstrated that EGCG induces epigenetic changes modulating hTERT activity through inhibition of DNMT and HAT activities Li and Tollefsbol ; Du et al.
All these data support the idea of EGCG as a key active nutrient for cancer inhibition through epigenetic control; however polyphenolic catechins generally exhibit poor oral bioavailability. Further investigation is required to improve EGCG absorption and metabolic biotransformation, in order to increase its potential effect in cancer prevention and therapy through epigenetic modulation.
Curcumin diferuloylmethane is a polyphenolic compound derived from turmeric Curcuma longa Linn with remarkable medicinal properties, mainly with anti-inflammatory and anti-cancer effects. Curcumin has been shown to modulate multiple intracellular pathways associated with proliferation, survival, invasion, apoptosis, and inflammation Park et al.
In silico molecular docking studies revealed that curcumin can block or inhibit the catalytic site of DNMT1, thus resulting in decreased enzymatic activity. In vitro experimental studies validated the DNMT1 and DNMT3B inhibition activity of curcumin in several human cancer cell lines Jiang et al.
Similarly, in vitro and in vivo experiments showed that curcumin and its synthetic analogue FN1 were able to restore the activity of Nrf2 gene by hypomethylation of its promoter and through inhibition of DNMTs activity, hence activating anti-oxidant pathways Li et al.
Several examples of phytochemicals involved in epigenetic modulation of DNA methylation dysregulation and chemoprevention in different carcinogenesis are presented in Table 1 , such as well-known resveratrol, quercetin and others. We will exemplify next with one phytochemical which exerts its epigenetic modulator capacity on various types of epigenetic alterations in carcinogenesis and could interfere with different layers of epigenetic mechanism, sulforaphane SNF.
Sulforaphane belongs to Brassicaceae family and represent the most effective chemopreventive agent among isothiocyanate ITC group of organosulfur compounds. Many studies have shown that SFN is an effective chemopreventive agent that has anti-proliferative, anti-inflammatory, anti-angiogenic, and anti-oxidative effects, as well as induction of differentiation, apoptosis, and cell cycle arrest in several types of cancers Cao et al.
SFN induces its chemopreventive effects partly by activation of phase I CYP enzymes and phase II detoxification enzymes, leading to restored mitochondrial function and reduced lipid peroxidation Kwon et al.
In human breast, colon and hepatocellular carcinoma, the chemopreventive activities of SFN are mediated, at least in part, through Nrf2 pathway activation, which modulates phase 2 detoxification enzymes, including NAD P H: quinone oxidoreductase 1 NQO1 and GST Cao et al.
A recent study demonstrates that SFN can activate the Nrf2 pathway in breast cancer cells, acting as an epigenetic modifier to regulate COMT expression to influence estrogen metabolism Cao et al. Numerous in vivo studies on murine models of colon, prostate, oral and pancreatic cancer showed the chemopreventive role of SNF by inhibiting tumor growth Hsu et al.
There has been an increased interest in SFN recently, due to its potency to influence epigenetic processes through targeting key epigenetic modulators such as DNA methyltransferases and HDACs, which may lead to local or global alterations of epigenetic hallmarks resulting in subsequent gene transcription and expression level changes Khan et al.
Also, SFN modulates DNA demethylation by downregulation of the expression of DNMT1 and DNMT3B, subsequently leading to induced demethylation of cyclin D2 gene promoter and expression in cancer cells Hsu et al. Similarly, in prostate cancer cells, SNF has been reported to be able to restore the expression of silenced GSTP1 by a mechanism involving promoter demethylation and increased histone acetylation.
These effects are associated with increased expression of the CDKNs p21 and p27, which are negative cell cycle regulators Hsu et al. Moreover, the SNF inhibition of the growth of prostate cancer PC-3 tumor xenografts could be correlated with inhibited HDAC activity.
In human subjects, a single dose of 68 g broccoli sprouts decreased HDACs activity significantly in peripheral blood mononuclear cells PBMC at 3 and 6 h following consumption Myzak et al. Recently, a comprehensive study of SNF chemopreventive effect on three breast cancer showed that SFN provoked cell cycle arrest and senescence are mediated by epigenetic changes, namely global DNA hypomethylation, decreased levels of DNMT1 and DNMT3B, and changes in microRNA profile in all studied cancer cells.
Moreover, SFN induced a decrease in m6A RNA methylation pattern that is also considered as an epigenetic regulation at the RNA level, recently discovered. Interestingly, in another study, authors demonstrated that SNF upregulates miR, which is a negative regulator of cancer stem cell formation in basal-like early stage breast cancer.
These results highlight its potential preventive properties for breast cancer Schnekenburger and Diederich SNF represents one example of bioactive molecules from natural sources that can modulate different epigenetic mechanisms in order to restore the normal function of genes involved in chemoprevention processes, as we show in Fig.
Sulforaphane modulates epigenetic mechanisms in chemoprevention. Sulforaphane induces activation of Nrf2 gene and upregulates the expression of anti-oxidative enzymes, related with cancer prevention mechanism.
Accumulating evidence suggests that the anti-cancer properties of sulforaphane could be at least partially mediated by its effect on epigenetic mechanisms.
Sulforaphane is a well-described DNMTs and HDACs inhibitor, reducing gene promoter-specific methylation and increasing total and promoter-specific histone acetylation in cancer cells.
Also, SFN can modulate the expression of several microRNAs, and at mRNA level it was associated with decreased 6-adenosine RNA methylation. To conclude, evidence-based data from both preclinical and clinical trials are now adding to support the benefits of bioactive compounds in preventing or mitigate tumor growth.
From the multiple processes altered in tumor cells, epigenetic ones, namely DNA methylation, can be modulated with the help of phytochemicals commonly found in natural foods and spices.
In support of such arguments, SNF holds a strong case, showing new beneficial sides of its well documented anti-oxidant activity. Whether these active compounds will continue to be only treatment adjuvants or will seize the lead in antitumor therapy, remains to be established by large cohort and epidemiologic studies.
Substantial experimental evidence indicates the potential importance of dietary and bioactive compounds in cancer prevention, but identifying direct relationships between diet and cancer in observational epidemiological studies and intervention trials had proven challenging.
Study design issues, imprecise dietary assessments, and a lack of consideration of tumor heterogeneity generally attenuate relative-risk estimates in observational studies; dietary biomarkers and characterization of etiological subtypes of cancers can help to better identify diet—cancer associations.
Epigenetic changes, such as DNA methylation, can be heritable, but are also influenced throughout life by environmental factors, such as diet, thus providing a novel avenue for lifestyle or therapeutic interventions.
Unlike the conventional drugs, phytochemicals have multiple targets and are thus of potential value in diseases like cancer, where multiple pathways are altered.
Moreover, they have selective toxicity targeting the cancer cells while showing negligible damage to normal cells. As demonstrated from taxol to sulforaphane, there is an unprecedented potential in exploring the herbal diversity for anti-cancer drug candidates.
DNMT inhibitors that are currently used in clinical trials are non-selective cytosine analogues with considerable cytotoxic side effects. Several natural products, such as EGCG, curcumin, sulforaphane, from diverse chemical classes, have shown DNMT inhibitory activity, but this property needs more in-depth investigations.
Subsequently, the membranes were incubated with a HRP-conjugated secondary antibody Protein Tech Group, Chicago, IL at room temperature for 1 h.
Detection was performed with the ECL kit GE Healthcare; Munich, Germany , according to the manufacturer's instructions.
Fluorometric assays of caspase activity were carried out by using the substrate Ac-DEVD-AMC BD Pharmingen, San Diego, CA for caspase 3 and Ac-IETD-AMC BD Pharmingen, San Diego, CA for caspase 8.
Briefly, cells were lysed in lysis buffer 10 mM HEPES, mM KCl, 5 mM MgCl2, 1 mM EDTA, 0. Following incubation for 30 min on ice, samples were centrifuged at 12, rpm for 30 min at 4°C and the protein content in supernatants was determined by Bradford dye method.
The release of the fluorogenic group AMC was determined at 37°C in a VersaFluor Fluorometer Bio-Rad, Hercules, CA with excitation at nm and emission at nm. Small interfering RNA siRNA for down-regulating XAF1 gene expression was done by transfection of RNA oligonucleotides with lipofectamine Invitrogen, USA according to the manufacturer's instructions.
One day before transfection, SW and LOVO cells were plated on a mm culture dish in RPMI complete medium. Briefly, cells were placed in 1 mL of siRNA mixture with nM siRNA and 5 µL lipofectamine After 8 h of transfection, 1 mL of RPMI complete medium was added, and experiments were conducted 48 h after transfection.
Protein levels were analyzed by Western blot. The negative control NC siRNA and siRNA against XAF1 were synthesized by Shanghai GenePharma Co. Total RNA was extracted with TRIzol Reagent Invitrogen, Carlsbad, CA, USA according to the manufacturer's instructions. cDNA was prepared from total RNA using random primers Promega, Madison, USA and the Omniscript RT kit Qiagen GmbH, Hilden, Germany.
The relative levels of mRNA were determined by real-time quantitative reverse transcription-PCR RT-PCR using an Eppendorf Realplex Mastercycler Eppendorf, Hamburg, Germany and Quantitect SYBR Green PCR kit Qiagen GmbH, Hilden, Germany.
Actin BioVision, Palo Alto, CA, USA mRNA levels were used for normalization. All experiments were repeated three times and were expressed as mean ± SD.
Statistical analysis was analyzed using the Statistical Package for Social Sciences SPSS software version To determine the effects of the combination treatment of DNA methyltransferase inhibitor decitabine and EGFR inhibitor gefitinib on human colon tumor cell viability, SW [27] and LOVO cells [28] carrying wild-type EGFR gene were exposed to different concentrations of decitabine or gefitinib alone or in combination for up to 48 h, followed by the determination of cell viability using MTT assay.
As shown in Fig. Additionally, Fig. Furthermore, Fig. Similarly, treatment of LOVO cells with fixed concentrations of decitabine decreased the IC 50 values of gefitinib from 5. A and B SW and LOVO cells were cultured in control conditions DMSO or in the presence of the indicated concentrations of decitabine DAC and gefitinib GEF , alone or in combination, for 48 h, and then assessed for viability by MTT assay.
Results are means of duplicate assessments from one out of three independent experiments. C and D SW cells and LOVO cells were plated, treated, and processed as in A and B.
Rectangle symbol and diamond symbol designate the CI value for each fraction affected effect. The effect ranges from 0 no inhibition to 1 complete inhibition. The data are representative of three independent experiments. E Influence of SW cells and LOVO cells on the number of colony-forming cells, as evaluated by clonogenic assay.
For colony-forming assay, the clonogenic assay was done as described in materials and methods. Columns, mean of three determinations; bars, SD. Results shown are representative of three independent experiments. These data suggested that the two compounds, decitabine and gefitinib, might synergize to inhibit cell viability in colon cancer cells.
To confirm this synergism, we treated cells with a combination of the two agents in a constant ratio to one another and used Calcusyn software to calculate the combination index CI following Chou and Talalay's method as described under Methods.
In addition, by clonogenic cell survival assay, we found that decitabine and gefitinib exerted synergistic effects to inhibit the clonogenic activity of SW and LOVO cells Fig.
Furthermore, the few cells surviving decitabine plus gefitinib generated colonies that were much smaller in size than those generated by cells surviving either of these agents alone data not shown. Notably, when used together, treatment of NCM cells, a normal human colon mucosal epithelial cell line, with decitabine and gefitinib showed an effect greater than when each compound was used individually, but effects were less than additive suggesting antagonism Fig.
S1A and B. Moreover, the combination of low concentration of decitabine 2. Meanwhile, we detected the cell viability of colon cancer cells treated using the two agents alone or in combination Fig.
S2B , and found that the combination of low concentration of decitabine and gefitinib did not significantly decrease cell viability. These results indicated that the reduction of cells migration caused by the two drugs was not involved in inhibition of cell viability. Decitabine and gefitinib significantly inhibited the growth of two types of colon cancer cells compared to the treatment with either agent alone.
As AKT and mTOR signaling pathways play a critical role in cell growth and cell apoptosis, we determined the effects of decitabine and gefitinib on the activation of these pathways.
We calculated CI values to further find that the combination of 10 µM decitabine with 5 µM gefitinib in SW cells or 4 µM gefitinib in LOVO cells was the most effective. SW and LOVO cells were treated with decitabine and gefitinib either alone or in combination.
After 48 h, the cells were processed for Western blot as described under methods. Additionally, we observed minimal reductions of phosphorylation of AKT, mTOR and S6K with 5 µM gefitinib in SW and 4 µM in LOVO cells. However, the combination of the two drugs completely abrogated AKT and mTOR activities in SW and LOVO cells Fig.
A and B SW and LOVO cells were plated, treated for 48 h with decitabine DAC and gefitinib GEF either alone or in combination, and the expression levels of AKT, mTOR, S6K, and phosphorylation were determined by Western blot analysis as described under Methods.
Expression of β-actin served as a loading control. To determine if the cytotoxic effects of gefitinib combined with decitabine were due to induction of apoptosis, SW and LOVO cells were treated with the two compounds, alone or in combination, for 48 h and then cell apoptosis was determined by Annexin V-FITC and propidium iodide PI staining and flow cytometry analysis.
However, there was a significantly higher apoptosis rate found upon treatment with the combination of gefitinib and decitabine Fig. Similar results were found in LOVO cells Fig. Additionally, cell apoptosis was measured by detecting sub-G1 population with PI staining and flow cytometry analyses.
S3 , the sub-G1 population percentages induced by the treatments with the two drugs combination were greater than those induced by the drugs individually. A and B SW and LOVO cells were cultured in control conditions DMSO or in the presence of the indicated concentrations of decitabine DAC and gefitinib GEF , alone or in combination, for 48 h.
And then cells were stained with Annexin V-FITC and propidium iodide PI and analyzed by flow cytometry. This experiment was done in triplicate and representative diagrams of Annexin V-FITC assays are shown. C Quantitative measurement of Annexin V-FITC flow cytometry analyses showed positive apoptotic cells in response to DAC and GEF, alone or in combination.
Columns, mean; bars, SD. D and E SW and LOVO cells were pre-treated with 10 µM z-VAD-fmk zVAD or 20 µM necrostatin-1 Nec1 or necrostatin-5 Nec5 for 1 h followed by treatment with the indicated concentrations of DAC and GEF, alone or in combination, for additional 48 h.
This experiment was repeated thrice. To determine whether or not gefitinib plus decitabine caused caspase cascade, the pan caspase inhibitor z-VAD-fmk 10 µM was used to pretreat SW or LOVO cells before treatment of gefitinib plus decitabine.
However, the cell apoptosis triggered by the two compounds in combination could not be blocked by necrostatin-1 or necrostatin-5, a novel class of potent small-molecule inhibitors of cell necrosis. These results revealed that the apoptotic pathway was involved in the cell death induced by gefitinib combined with decitabine, in colon cancer cells.
Since apoptosis is tightly regulated by pro- and antiapoptotic members of the Bcl-2 protein family, the proapoptotic factors BAX, BID and BIM as well as the antiapoptotic proteins Bcl-2 and Bcl-XL were studied in SW and LOVO cells after treatment with decitabine or gefitinib or their combination by Western blot analysis.
SW and LOVO cells responding to decitabine plus gefitinib manifested increased amounts of the proapoptotic BAX as well as major reduction in the levels of the anti-apoptotic protein Bcl-2 Fig. However, the two compounds in combination failed to affect the expression levels of other members of the Bcl-2 protein family, including the proapoptotic proteins BID and BIM as well as antiapoptotic protein Bcl-XL data not shown.
SW and LOVO cells were plated, treated for 48 DAC and gefitinib GEF either alone or in combination. A and B the expression levels of cleaved-caspase 3, cleaved-PARP, XAF1, XIAP, BAX, Bcl-2 were determined by Western blot analysis as described under Methods.
C and D The caspase 3 activity was quantified as described under methods. E and F The caspase 8 activity was quantified as described under Methods. Inhibitor of apoptosis protein IAP is a protein family acting through the inhibition of caspase activity.
The X-linked IAP XIAP , a member of IAP, and XIAP-associated factor 1 XAF1 were examined in colon cancer cells stimulated with decitabine along with gefitinib. Notably, combined treatment with both drugs remarkably increased the expression of XAF1 compared to single agent treatment Fig. To test the ability of gefitinib combined with decitabine to activate caspases, cleaved caspase 3 and cleaved PARP were studied in SW and LOVO cells after treatment with gefitinib or decitabine or their combination by Western blot analysis.
Significant increase in the amounts of both cleaved caspase 3 and cleaved PARP were noted in colon cancer cells treated with two drugs in combination compared to treatment with single agents Fig.
Moreover, colon cancer cells treated by the two compounds were analysed for caspase 3 and caspase 8 activities by fluorogenic substrate cleavage. Additionally, decitabine plus gefitinib exerted time-dependent caspase 3 activity inductions on SW and LOVO cells Fig.
In contrast, no changes were found in caspase 8 activities Fig. The data shown above indicated that decitabine combined with gefitinib increased the XAF1 levels in SW and LOVO cells. We then asked if the two drugs in combination could enhance XAF1 mRNA levels in colon cancer cells.
Furthermore, colon cancer cells were treated with the two drugs in combination for different time intervals. We showed that XAF1 protein and mRNA levels were increased by decitabine plus gefitinib in a time-dependent manner Fig.
A and B SW and LOVO cells were treated for 48 h with the indicated concentrations of decitabine DAC and gefitinib GEF either alone or in combination.
The mRNA expression levels of XAF1 were determined by real-time quantitative PCR. Expression of β-actin served as control. C and D SW and LOVO cells were treated for the indicated time intervals with the indicated concentrations of DAC and GEF in combination.
The expression levels of XAF1 were determined by Western blot analysis as described under methods. E and F SW and LOVO cells were treated for the indicated time intervals with the indicated concentrations of DAC and GEF in combination. G SW and LOVO cells were treated with nM XAF1 siRNA and negative control NC siRNA for 12 h, and treated with the indicated concentrations of DAC and GEF in combination for an additional 48 h.
The knockdown effects on XAF1 were confirmed by Western blot analysis right panel. To more directly assess the role of XAF1 in the apoptotic activity of the decitabine combined with gefitinib, cell apoptosis was evaluated in colon cancer cells treated with the two drugs in combination with siRNA-mediated knockdown of XAF1.
Together, these findings suggested that XAF1 contributed to the sensitivity of colon cancer cells to apoptotic induction after combined treatment with gefitinib and decitabine. There have been multiple investigations of chemotherapeutics that target EGFR and thereby attenuating the EGFR signaling pathway in colon cancer [29].
However, response effects for single EGFR inhibitor remain relatively modest unless the EGFR-targeted therapy is combined with other chemotherapeutics [30] , [31] , [32] , [33]. Consistent with previous findings, we found that EGFR inhibitor gefitinib alone modestly decreased the cell viability in SW and PC-9 colon cancer cell lines carrying wild-type EGFR gene [27] , [28].
DNA methyltransferase inhibitors as promising anti-tumor agents have been shown to demethylate and upregulate the expression of tumor suppressor genes and display strong anti-tumor growth effect in vitro , in vivo and in the clinic [34]. Although DNA demethylating agents induced apoptosis are minimal on its own, they exert potential to enhance the effects of other chemotherapeutics, such as DNA damaging agent cisplatin [35].
In the current study, decitabine combined with gefitinib was more effective to inhibit cell viability than single agent alone in colon cancer cells. Combination index data analysis showed that this combination is highly synergistic at inhibiting cell viability of colon cancer cells. Clonogenic assay results showed the number of positive colonies was strikingly reduced in the cells treated with the combination therapy, suggesting that the damage inflicted by the interaction of treatments was chronic and that the affected cells were not able to recover.
Furthermore, Annexin V assay showed that the combination of decitabine plus gefitinib was synergistic at inducing apoptosis in colon cancer cells. These results raised the possibility that the combination of decitabine and gefitinib exerted a dual anticancer action in colon cancer cells, which consisted in cell apoptosis induction plus cell-cycle blockade.
Additionally, the combination was synergistic at inhibiting colon cancer cells migration. More importantly, the combination of decitabine and gefitinib displayed minimal toxicity to NCM cells, a normal human colon mucosal epithelial cell line.
The data shown above indicated a novel therapeutic strategy using a combination of decitabine and gefitinib in human colon cancer. AKT has been shown to directly interact with and phosphorylate XIAP [39] , [40].
XIAP phosphorylated by AKT can prevent XIAP degradation and thus inhibit caspase 3 activation confer resistance to apoptosis [41]. Therefore, we examined the expression of XIAP in colon cancer cells treated by the two agents alone or in combination.
Unexpectedly, we did not detect an obvious reduction of XIAP expression in the colon cancer cells treated by two compounds in combination. One possible explanation for the discrepancy of our results and previous studies might be due to the interaction between XIAP and AKT was in specific histologic types of cancer or in cell line specific pathways.
Several studies indicated that cell cycle regulatory gene CDKN2A is hypermethylated in colon cancer cells [42] , and decitabine-induced cell proliferation inhibition may result from the release of methylation silencing of the CDKN2A gene [43].
In addition, previous studies suggested that allelic loss of the XAF1 gene is prevalent in cancer cell lines [44]. In contrast, XIAP levels are relatively high in the majority of cancer cell lines [45].
Studies suggested that a high level of XIAP to XAF1 expression in cancer cells may provide a survival advantage through the relative increase of XIAP anti-apoptotic function [46] , [47]. Moreover, in many colon cancers, the CpG island of XAF1 gene is hypermethylated, resulting in transcriptional repression [48] , [49] , [50].
In the present study, the expression of XAF1 was induced by DNA demethylating agent decitabine in SW and LOVO cells, raising a possibility that decitabine increased XAF1 level by preventing the CpG island methylation of XAF1 gene.
More importantly, the expression of both mRNA and protein levels of XAF1 was remarkably increased in colon cancer cells treated by using two drugs in combination compared to single agent treatment.
Gefitinib is known to inhibit transmembrane transporters of the ABC family, including the P-gp, MRP1 and BCRP [51]. After cellular uptake, the first limiting step is the ATP-dependent phosphorylation of nucleosides to form monophosphorylated nucleotides [ 36 ].
These monophosphorylated nucleotides are incorporated into DNA in the place of cytosine. Then, DNMTs recognize the azacytosine-guanine dinucleotide and catalyze the methylation reaction by forming a covalent bond with the cytosine ring [ 37 ]. The covalent complex at C6 cannot be resolved through b-elimination, because of the presence of a nitrogen atom at position 5.
Covalently trapped DNMTs are degraded, resulting in the depletion of cellular DNMTs [ 36 , 38 ]. High-dose DNMT inhibitors facilitate the formation of bulky adducts, leading to replication fork stalling and DNA replication inhibition, which causes cell death [ 39 ].
When cells are treated with low DNMT inhibitor doses, the agents are still incorporated into DNA and bind DNMTs, leading to DNMT degradation. Without DNMTs to maintain DNA methylation, CpG sites lose their methylation after cell replication, and the transcription of genes previously silenced by promoter methylation is restored [ 40 , 41 ].
Decitabine can decrease DNMT1 and DNMT3A expression, reversing abnormal transcription activation, while azacitidine only targets DNMT1 [ 42 ]. Another difference between these two drugs is that azacitidine can be incorporated into both DNA and RNA, whereas decitabine can only be incorporated into DNA [ 18 ].
Azacitidine, an analog of the cytidine pyrimidine nucleoside, has received approval by the US Food and Drug Administration for the treatment of all subtypes of MDS [ 43 ].
Despite marked activity in myeloid malignancy, the use of azacitidine in patients with solid tumors is limited by toxicity, myelosuppression; and low complete and partial response rates Table 1 [ 44 , 45 ].
Recently, a two-part phase I study evaluated CC an oral formulation of azacitidine in combination with cytotoxic agents or as monotherapy for patients with advanced solid tumors. CC monotherapy resulted in partial responses three of eight patients and stable disease four of eight patients in patients with nasopharyngeal cancer.
Considering the potential benefit of CC as monotherapy in this study, the combination of CC with immune checkpoint inhibitors could be a promising area of clinical investigation [ 46 ]. Decitabine is a unique cytosine analog and has recently emerged as a therapy for MDS and CML. Although the promise of these hypomethylating drugs has not been realized for solid tumor cancer therapy, researchers contend that decitabine can achieve optimal biological effects at low doses [ 47 ].
In the s, decitabine monotherapy produced unsatisfactory results for patients with solid tumors [ 48 , 49 ]. After the treatment cycles, no objective responses were observed, and seven of ten patients exhibited disease progression after one or two cycles.
Samlowski et al. Zebularine is a cytidine analog that lacks the amino group at position 4 of the pyrimidine ring. Zebularine has high stability and low toxicity, and it is stable at acidic and neutral pHs, enabling oral administration [ 37 , 50 ].
When zebularine traps DNMT on DNA, zebularine becomes an obstacle for the second round of replication. This results in a collapsed replication fork and the formation of replication-dependent double stand breaks DSBs [ 51 ]. Moreover, zebularine can suppress the interaction of DNMT1 with G9a histone methyltransferases, which may regulate the survival and apoptosis of human cancer cells [ 52 ].
Transient zebularine exposure produces differential cell density-dependent responses and correlates with the overexpression of genes related to cancer stem cells and the key epithelial—mesenchymal transition process [ 53 ].
Although zebularine is more stable and less toxic than azacitidine and decitabine, clinical trials are required to demonstrate its therapeutic effect in solid tumors. Guadecitabine SGI is a second-generation decitabine and deoxyguanosine compound with prolonged half-life and activity in AML and high-risk AML.
Guadecitabine addresses the shortcomings of first-generation DNMT inhibitors that are susceptible to deamination by cytidine deaminase CDA. CDA is found in multiple organs in the body, causing first-generation DNMT inhibitors to have short plasma half-lives.
Guadecitabine has improved stability that confers enhanced DNA incorporation into dividing cells and is more resistant to CDA [ 54 ]. Based on these factors, it is believed that guadecitabine may be a more appropriate DNMT inhibitor than azacitidine and decitabine [ 55 , 56 ].
Guadecitabine has been demonstrated to have clinical activity in MDS and AML [ 57 , 58 ]. However, a substantial difference in cost in combination with a marginal difference in survival benefit might limit its use in the clinical setting [ 59 ]. This compound incorporates into the DNA sequence recognized by the bacterial C5 DNA methyltransferase M.
In both in vitro and in vivo models, TdCyd and FdCyd potently deplete DNMT1 in cancer and concomitantly inhibit tumor growth [ 63 ]. To overcome the disadvantages of nucleoside analogs, including poor bioavailability, chemical instability under physiological conditions and a lack of selectivity, nonnucleoside analogs have been developed over the last decades [ 64 ].
The structures of nonnucleoside analogs are very heterogeneous, but their mechanisms of action are independent of DNA incorporation.
Some drugs including procainamide, an amide, and its ester analog procaine have been repurposed after they were shown to have demethylating effects. These agents show affinity for CpG-rich regions of DNA, blocking the activity of DNMTs and reactivating some tumor suppressor genes [ 65 ].
SGI was synthesized as a quinoline-based compound and was described for against DNMT1, DNMT3A and DNMT3B [ 66 , 67 ]. After that, Valente et al. SGI and its analogue share DNA-competitive and AdoMet non-competitive behavior on DNMT1 [ 64 ].
SGI may inhibit DNMT activity, induce the degradation of DNMT1 and reactivate tumor suppressor genes [ 69 ]. SGI can also impair cervical cancer cell and hepatocellular carcinoma cell propagation by dramatically increasing apoptotic cell death and cell cycle arrest [ 69 , 70 ].
As a novel DNMT inhibitor, MC is more potent and selective than SGI toward other S-adenosylhomocysteine-dependent SAM-dependent methyltransferases [ 71 , 72 ].
Zwergel et al. reported that MC displays a stronger in cell demethylating ability than both azacitidine and decitabine.
Besides, this compound proved antiproliferative activity in several cancer cell line types [ 73 ]. In addition to SGI, some oligonucleotides are accommodated in the catalytic pocket of DNMTs, where they effectively function as competitive inhibitors.
MG98 has shown interesting preclinical evidence that it can inhibit DNMT1 [ 74 ], allowing for the re-expression of tumor suppressor genes and tumor growth inhibition [ 75 , 76 ]. In an open-label phase I study, patients with advanced solid malignancies were treated with escalating MG98 doses administered as a continuous infusion over 7 days repeated every 14 days.
After two cycles, suppression of DNMT1 expression was observed in 26 of the 32 patients studied. One patient achieved a partial response, and another achieved prolonged disease stabilization [ 76 ]. N-Phthaloyl-L-tryptophan RG , a DNMT1 inhibitor [ 77 ], targets DNMT1 SAM cofactor binding.
Nanaomycin A is the first selective DNMT3B inhibitor that can induce genomic demethylation. Nanaomycin A interacts with DNMT3B amino acid residues that are involved in methylation, preventing DNMT3B from participating in normal DNA methylation [ 81 ].
Nanaomycin A treatment reduces global methylation levels in cancer cell lines and reactivates transcription of the RASSF1A tumor suppressor gene [ 82 ]. Kumar et al. Moreover, γ-radiation increased the transcriptional activity of the p16 INK4a and ATM gene promoters by altering DNA methylation levels.
Together with In-DTPA-hEGF, decitabine can sensitize breast cancer to ionizing radiation and induce DNA destruction [ 84 ]. Kim et al. After this treatment, colon cancer cell growth was significantly lower than that with decitabine or radiotherapy alone, and increases in the number of G1-phase cells and the apoptosis rate were observed for colon cancer cells.
Recently, Ou et al. In the s, Fost. et al. They demonstrated the synergistic cytotoxicity of this drug combination against a panel of six human cell lines. Epigenetic priming with decitabine can improve the sensitivity of gastric cancer cells to SN38 doxorubicin and cisplatin [ 88 ].
Low-nanomolar doses of decitabine and azacitidine induce sustained antitumor responses [ 89 ]. In myeloma cell lines, researchers observed a significant phenomenon of cell proliferation inhibition after combination therapy of decitabine with adriamycin [ 90 ]. Several studies have investigated the molecular mechanisms through which DNMT inhibitors affect the efficacy of other drugs Fig.
Molecular regulatory mechanisms of DNMT inhibitors in increasing the sensitivity to drugs. DNMT inhibitor treatment can increase the sensitivity of chemotherapeutic drugs via the methylation status of ARNTL, RASS1, MLH1, hMLH1, WT1 and BCL DNMT inhibitors are able to sensitize tumor-targeting drugs through the induction of various proteins, such as EREG, EGFR and XAF1.
They can also enhance immunotherapy by targeting EZH2 and MAGE A comparative study showed that platinum-resistant cell lines exhibited more epigenetic alterations than platinum-sensitive cell lines, and the hypermethylation of promoter regions was significantly increased.
The authors identified 14 genes that were hypermethylated in cisplatin-resistant cell lines but not in cisplatin-sensitive parental cell lines. Six of 14 genes SAT, C8orf4, LAMB3, TUBB, G0S2 and MCAM were cisplatin inducible in sensitive cell lines but not in resistant cell lines [ 91 ]. DNMT inhibitors demethylated the promoter CpG regions of ARNTL.
The ARNTL protein suppressed NPC cell proliferation and enhanced cell sensitivity to cisplatin by targeting CDK5.
ARNTL overexpression suppressed NPC cell proliferation in vitro and in vivo, and the opposite effect was observed following ARNTL silencing.
Gene set enrichment analysis GSEA revealed that ARNTL is associated with the cell cycle and that ectopic expression and overexpression of ARNTL could induce G2-M phase arrest [ 92 ].
Moreover, in an in vivo melanoma model, DNMT inhibitors augmented the hypermethylation status of the RASSF1 gene promoter, targeted the CTGF and CYR61 genes through the hippocampal pathway and increased the sensitivity of bladder cancer cells to cisplatin and adriamycin [ 15 ].
Moreover, MLH1 expression was closely related to the methylation status of the hMLH1 promoter [ 93 ]. Several studies have shown that decitabine can reverse cisplatin resistance by inhibiting hMLH1 in human non-small cell lung cancer NSCLC and esophageal carcinoma [ 95 , 96 ].
VHL-TGFBI hypomethylation was found to be related to the sensitivity to paclitaxel PTX [ 99 ]. Methylation of the EGFR promoter inhibits EGFR expression in a variety of tumor cells. Three NSCLC cell lines H, H and PC-9 with different EGFR mutation statuses and levels of EGFR-TKI sensitivity were used in this study.
The results showed that the EGFR promoter region was unmethylated in PC-9 cells and that these cells were sensitive to gefitinib an EGFR-TKI drug. In contrast, the EGFR promoter region was methylated in H and H cells, and the cells were resistant to gefitinib [ ].
Treatment with decitabine resulted in the re-expression of EGFR in CAMA1 and MB cell lines, which are relatively resistant to gefitinib. However, after cotreatment with decitabine and gefitinib, a significant effect was observed on apoptosis induction.
DNMT inhibitors can reverse the hypermethylation status of EGFR promoters in different cancers, which may enhance EGFR expression and reverse EGFR-TKI resistance [ 20 , 21 , ]. Qu et al. Jiyoeu et al. DNMT3b knockdown significantly increased EREG expression and did not significantly affect EREG promoter methylation.
In another study, combined treatment with decitabine and gefitinib increased XIAP-associated factor 1 XAF1 expression, which plays an important role in apoptosis [ ]. Significantly tumor growth inhibition and prolonged survival were observed in the CT26 mouse model after treatment with a combination of PD-1 blockade and decitabine versus treatment with decitabine or PD-1 blockade alone.
Decitabine may provide clinical benefits to patients with colorectal cancer and low microsatellite instability or microsatellite stability [ ]. In NSCLC, combining the DNA hypomethylating agent azacytidine with anti-PD-1 therapy significantly reduced tumor size compared with that with anti-PD-1 therapy alone.
This combination might therefore be a promising approach to overcoming anti-PD-1 resistance [ ]. High levels of methylated CFTR are observed in breast cancer, and CFTR overexpression can inhibit breast cancer cell growth. Increased cell invasion was observed following CFTR knockdown. These results suggest that CFTR might be a diagnostic marker of breast cancer [ ].
DACT2 is frequently inactivated by CpG methylation in NPC. A study suggested that DACT2 promoter methylation was a potential epigenetic biomarker for the detection of NPC and for chemotherapy guidance [ 96 ]. Stewart et al. This study implicated KRAS status as a biomarker of drug response in ovarian cancer.
BRAF Ve plays an important role in melanoma tumorigenesis. Hou et al. The results indicate that a wide range of genes with broad functions are linked to BRAF VE signaling through hypermethylation or hypomethylation. Low-dose decitabine treatment remarkably enhanced the effects of cisplatin and gemcitabine on basal-like bladder cancer in vivo and in vitro.
These effects were accompanied by decreases in genome-wide DNA methylation, gene re-expression and changes in key cellular regulatory pathways, including STAT3 signaling [ ].
DNA methylation status sequencing at different time points during colitis-associated cancer CAC revealed that genes were hypermethylated at different time points during CAC initiation and progression.
Tumor growth and drug response were assessed in PANC-1 cells pancreatic ductal adenocarcinoma, PDAC after exposure to a noncytotoxic dose of azacitidine. The authors observed that unique peptides SST and SSTR2 were expressed in the pancreas and confirmed that azacitidine epigenetically reprogrammed PANC-1 cells to induce anticancer effects [ ].
DNMT inhibitors promoted MIG-6 re-expression by inhibiting MIG-6 promoter methylation. The negative feedback of MIG-6 expression increased the number of EGFR receptors [ ]. Chou-Talalay analysis showed that, in bladder cancer cells, the combination of decitabine with an entinostat ENT histone deacetylase inhibitor could not reverse chemoresistance.
However, the combination treatment between decitabine and ENT led to forkhead box class O1 FoxO1 upregulation, and FoxO1 expression resulted in increased relapse-free survival in patients with bladder cancer.
Moreover, this combination further activated proapoptotic Bim and p21, cell cycle regulators [ ]. These results show that low FoxO1 expression in tumor specimens may be associated with resistance to cisplatin first-line therapy in patients with bladder cancer.
The immune system maintains the function of the body when attacked by external substances through its two roles as a "monitor" and "protector" [ , ]. Deregulated immune systems cannot effectively kill tumor cells, leading to immune evasion [ ].
There is evidence that tumor immune evasion is mediated by nonmutational epigenetic events involving chromatin and that epigenetics and mutations collaborate to determine the state of tumor progression. Although human endogenous retroviral sequences ERVs make up approximately 8.
Several studies have highlighted the importance of DNA methylation in the suppression of ERVs [ ]. It is possible that DNMT inhibitors can reactive ERVs. After reactivation, repeat elements produced by ERVs may form nucleic acid molecules of various configurations that are then sensed by the innate immune machinery to trigger an immune response [ ].
Decitabine treatment may result in the production of the antigen encoded by MAGE-1 a cancer testis antigen CTA member. MAGE-1 is associated with major histocompatibility complex class I molecules at the cell surface for T-cell recognition [ ]. Thus, CTAs are a potential source of new tumor cell surface antigens and are widely used in CAR T cell production [ , ].
The efficacy of coupling an immune checkpoint blockade approach with a DNMT inhibitor may be increased by taking advantage of a bystander effect by attracting T cells to the tumor and simultaneously enforcing the uniform expression and display of CTAs [ ].
DNA hypomethylation directly enhances PD-L1 expression in tumor cells and increases the expression of immune-related genes and T cell infiltration [ ]. Overexpression of DNMT1 and EZH2 can result in the consumption of B cells and prevent macrophage production.
This may explain why decitabine can increase the antitumor T cell response [ ]. In another study, Peng et al. announced that DNMT inhibitors may improve the clinical efficacy of MAGE-A3-specific T cell therapy by increasing target gene expression [ ].
The majority of combination DNMT inhibitor therapies assessed to date have involved the combination of decitabine and platinum drugs. We collected decitabine-based clinical trials from the National Center for Biotechnology Information NCBI database in April Table 2. In , Schwartsmann et al. However, only a short-lasting partial response was observed in a single patient with cervical cancer, and two minor responses were documented in patients with NSCLC and cervical cancer [ ].
Pohlmann et al. also reported the administration of a decitabine-based combination in Evaluation after 2 cycles revealed a satisfactory response rate, with eight patients Patients with ovarian cancer are often administered a platinum compound and a taxane.
Several phase I or phase II clinical trials used a low dose of decitabine combined with carboplatin to treat platinum-resistant ovarian cancer or relapsed ovarian cancer Table 2 [ , , , , , ].
This approach achieved an effective clinical response, with nine patients Low-dose decitabine altered gene DNA methylation and cancer pathways, restored carboplatin sensitivity in patients with heavily pretreated ovarian cancer and resulted in a high objective response rate and prolonged progression-free survival [ ].
A phase I clinical trial recruited pediatric patients with solid tumors. In another phase I study, Stathis et al. studied different doses of decitabine and vorinostat six sequential and three concurrent doses.
The results showed that 11 of the 38 patients with solid tumors and non-Hodgkin's lymphoma had a stable response after four treatment cycles [ ].
Garrido-Laguna et al. conducted a phase I study to evaluate decitabine in combination with panitumumab an antibody against EGFR in wild-type KRAS metastatic colorectal cancer mCRC patients. Ten patients had stable disease three of them had stable disease longer than 16 weeks.
Decreased MAGE promoter methylation was not observed in peripheral blood mononuclear cells [ ]. The BRAF gene regulates the methylation of a wide number of genes and affects multiple cellular functions [ ]. Fourteen V E BRAF-positive patients with metastatic melanoma were placed into four groups, and each group received a different regimen.
Three patients achieved a complete response, three had a partial response, and five had stable disease. Preclinical assessment demonstrated that this combination treatment delayed the development of acquired resistance and improved the duration of treatment sensitivity [ ].
In NSCLC, immunotherapy produced an astounding result. An objective response a complete or partial response was observed in 5 of 49 patients with NSCLC. These patients passed the week point without progression with subsequent immune checkpoint therapy, and three of the five developed high-grade partial responses according to the Response Evaluation Criteria in Solid Tumors RECIST that remained durable over 2.
Two different clinical trials combined decitabine and cytokine-induced killer CIK cells. The first study divided 52 recurrent ovarian cancer patients with platinum resistance into two groups. Patients in the paclitaxel and carboplatin DTC group were treated with decitabine and a reduced dose of paclitaxel and carboplatin.
Patients received decitabine on days 1—5 and were then divided into two groups. The toxicity and overall response rate observed did not significantly differ between cancer types and treatment cohorts.
The mechanism by which DNMT inhibitors function in combination with antitumor drugs has not yet been fully elucidated. However, the studies explored in this review show that, in most cases, combination treatment with DNMT inhibitors and antitumor drugs has higher efficacy than treatment using antitumor drugs alone.
However, there are many hurdles to overcome before the routine clinical application of this therapeutic approach. The sample size for clinical trials is small, with most studies involving fewer than 50 patients.
Moreover, there are very few studies that use randomized, blind, controlled designs. Although combination treatments using DNMT inhibitors and antitumor drugs may provide helpful insights into the development of efficient therapeutic approaches for cancer treatment, further investigation is needed.
Such studies should include randomized controlled trials with large sample sizes. Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. Riley RS, June CH, Langer R, Mitchell MJ.
Delivery technologies for cancer immunotherapy. Nat Rev Drug Discov. Article CAS PubMed PubMed Central Google Scholar. Fesnak AD, June CH, Levine BL. Engineered T cells: the promise and challenges of cancer immunotherapy.
Nat Rev Cancer. Antonarakis ES, Piulats JM, Gross-Goupil M, et al. Pembrolizumab for treatment-refractory metastatic castration-resistant prostate cancer: multicohort, open-label Phase II KEYNOTE study. J Clin Oncol. Article CAS PubMed Google Scholar.
Le DT, Kim TW, Van Cutsem E, et al. Chan TA, Glockner S, Yi JM, et al. Convergence of mutation and epigenetic alterations identifies common genes in cancer that predict for poor prognosis. PLoS Med. Tsai H-C, Baylin SB. Cancer epigenetics: linking basic biology to clinical medicine.
Cell Res. Goll MG, Bestor TH. Eukaryotic cytosine methyltransferases. Annu Rev Biochem. Zhang W, Xu J. DNA methyltransferases and their roles in tumorigenesis. Biomark Res. Article PubMed PubMed Central Google Scholar. Jeltsch A. Molecular enzymology of mammalian DNA methyltransferases.
Curr Top Microbiol Immunol. Schübeler D. Function and information content of DNA methylation. Zhang J, Yang C, Wu C, Cui W, Wang L.
DNA methyltransferases in cancer: biology, paradox, aberrations, and targeted therapy. Article CAS PubMed Central Google Scholar. Kim M, Costello J. DNA methylation: an epigenetic mark of cellular memory. Exp Mol Med. Sato T, Issa J-PJ, Kropf P. DNA hypomethylating drugs in cancer therapy.
Cold Spring Harb Perspect Med. Article CAS Google Scholar. Ahuja N, Sharma AR, Baylin SB. Epigenetic therapeutics: a new weapon in the war against cancer.
Annu Rev Med. Khandelwal M, Anand V, Appunni S, et al. Decitabine augments cytotoxicity of cisplatin and doxorubicin to bladder cancer cells by activating hippo pathway through RASSF1A. Mol Cell Biochem. Pinto A, Maio M, Attadia V, Zappacosta S, Cimino R.
Lancet Lond Engl. Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia.
Momparler RL. Pharmacol Ther. Nie J, Liu L, Li X, Han W. Decitabine, a new star in epigenetic therapy: the clinical application and biological mechanism in solid tumors.
Cancer Lett. Montero AJ, Díaz-Montero CM, Mao L, et al. Epigenetic inactivation of EGFR by CpG island hypermethylation in cancer. Cancer Biol Ther. Micevic G, Theodosakis N, Bosenberg M. Aberrant DNA methylation in melanoma: biomarker and therapeutic opportunities.
Clin Epigenet. Olino K, Park T, Ahuja N. Exposing hidden targets: combining epigenetic and immunotherapy to overcome cancer resistance. Semin Cancer Biol. Topper MJ, Vaz M, Marrone KA, Brahmer JR, Baylin SB. The emerging role of epigenetic therapeutics in immuno-oncology.
Nat Rev Clin Oncol. Article PubMed Google Scholar. Ehrlich M, Gama-Sosa MA, Huang LH, et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.
Nucleic Acids Res. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev. Lister R, Pelizzola M, Dowen RH, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R.
Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci USA. Role of CpG context and content in evolutionary signatures of brain DNA methylation.
Cheishvili D, Boureau L, Szyf M.
Editor-in-Chief: The importance of breakfast for teens cancet, FRS Honorary Life Czncer Kings College University preventikn Cambridge Cambridge UK. Non-GMO chips Print : ISSN Online : X. Page: [ - ] Pages: DOI: It is well established that aberrant gene regulation by epigenetic mechanisms can develop as a result of pathological processes such as cancer. The importance of breakfast for teens inhibitor High-potency natural fat burner about PLOS Subject Areas, Managing blood sugar during fasting here. Methylwtion recent advances in the treatment of human preventkon cancer, preventiin chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the canceer growth factor receptor EGFR and DNA methyltransferase inhibitosr examined in human colon cancer cells. We demonstrated that decitabine a DNA methyltransferase inhibitor synergized with gefitinib an EGFR inhibitor to reduce cell viability and colony formation in SW and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM cells, a normal human colon mucosal epithelial cell line. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor.Epimutations, Methylatjon as the unhibitors and epigenetic silencing of tumor suppressor genes, play a inhbiitors in the etiology of human Blood pressure regulation. In contrast to DNA mutations, which are passively inherited through Inhiibitors replication, epimutations must Muscle development diet actively maintained because csncer are reversible.
In fact, the cance of ibhibitors by small-molecule inhibitors inhibitorss the foundation for the orevention of cqncer inhibitors in novel cancer therapy strategies. EMthylation the compounds that ;revention epigenetic processes, prefention most extensively fr are Methjlation methyltransferase csncer.
In this review, we examine the literature on DNA methyltransferase inhibitogs and discuss knhibitors efficacy of such compounds as antitumor agents, as evaluated in phase I—III clinical Mrthylation. We also discuss future areas of research, including the development inhibitofs nonnucleoside Monitoring sodium levels, the application of novel bioanalytical tools for DNA methylation analysis which will be prevenhion for High-potency natural fat burner clinical ijhibitors of these compounds by allowing rational approaches pprevention trial Fast fat burningthe need to High-potency natural fat burner treatment schedules for maximal biologic effectiveness, and the need to define inhibktors endpoints High-potency natural fat burner pevention changes Methyaltion by demethylating drugs inhbiitors patients can be monitored during treatment.
Assays for Metjylation and preveniton DNA Methylatiom also need cancee be further inhjbitors to establish the pharmacodynamic parameters of DNA methyltransferase inhibitors preventjon patients and to provide rational orevention to maximizing the therapeutic efficacy of these compounds.
Epigenetic mechanisms regulate the orevention of genetic information. Epigenetic High-potency natural fat burner of DNA fpr histones are stable Metabolism boosting supplements heritable fkr are also reversible 1.
They include covalent fog of bases in the DNA and of amino acid prevenntion in the histones. DNA methyltransferases are a family of preevntion that methylate DNA at the carbon-5 position of cytosine residues 2.
Methylated DNA can then be bound by methyl-binding proteins 3 inyibitors function as adaptors between inhbiitors DNA and chromatin-modifying enzymes Methylatkon.
Histone-modifying enzymes then covalently Bitter orange for energy the amino-terminal residues of histones to induce the formation of chromatin structures that repress gene Methhlation 4. Examples of covalent histone modifications Methylation inhibitors for cancer prevention the methylation of the lysine at position prevdntion in histone H3 and the deacetylation prevenyion the lysine at position 16 in histone H4, both of which are associated with gene silencing.
Methulation interplay between DNA methylation and histone modifications has a profound effect on the epigenetic regulation of gene Anti-aging skincare patterns 5 and can thus become an Methylztion factor in the development of cancer, Methylation inhibitors for cancer prevention.
Epigenetic regulation by DNA methyltransferases methyl-binding proteins and histone modifying enzymes. DNA is methylated by DNA canced DNMTs. Methylated cytosine Methylatio solid circles are bound by methyl-binding cancwr MBPs Methylatipn subsequently recruit histone deacetylases HDACs and histone methyltransferases HMTs.
These Methylatkon mediate complex changes in the histone The importance of breakfast for teens pattern of methylated genes that result Methykation the establishment of repressive chromatin structures. DNA methylation is a crucial Methylation inhibitors for cancer prevention associated with epigenetic regulation.
Prevehtion methylation triggers chromatin reorganization that is High-potency natural fat burner by methyl-binding Methhlation Fig. Changes in the Mwthylation of DNA methylation, Methylarion increased hypermethylation or decreased Methypationhave pevention identified in all pevention of cancer cells examined so far.
In addition, it inhiibitors also been shown that genetic lesions in Methylatkon cancer cells can promote epigenetic alterations.
The leukemia-promoting PML—RAR Methylwtion protein, for example, can prevengion DNA methyltransferases to the target genes for the fusion protein and thereby inhibtiors epigenetic silencing 6.
These results provided an important paradigm for MMethylation cooperation of genetic and epigenetic lesions in promoting tumorigenesis. Genomic tumor Preventiob is generally characterized by preventtion methylation changes that inhiibtors also been prsvention epimutations.
At Mtehylation global level, the DNA is often ffor, particularly preventiion centromeric repeat sequences, and this csncer has been linked to genomic instability 7.
Another class of epimutations is characterized by the local hypermethylation of individual genes, which ffor been associated with aberrant gene silencing 8. Epimutations have been described in many types of prevemtion and appear to play an important role in tumorigenesis.
For Metyhlation, epigenetic Fiber-rich foods for digestion, rather than gene mutation, is Essential nutrient absorption main mechanism of Herbal remedies for natural pain management of the DNA High-potency natural fat burner repair gene hMLH1 Methylahion sporadic colon cancer canceeand the methylation of E-cadherin has a central role in metastasis and invasion of breast cancers Such epimutations rarely canxer in healthy tissue, ;revention that epigenetic therapies may have Balancing dietary needs tumor specificity.
The reversibility of prevenntion modifications Nutrient timing for nutrient timing for nutrient distribution them attractive Coenzyme Q and eye health for therapeutic interventions In contrast prrvention genetic mutations, which are fro passively through DNA replication, epigenetic gor must be actively maintained.
Consequently, pharmacologic prevwntion of certain Methylwtion modifications could correct Respiratory health news modification patterns and prevenhion directly change Methjlation expression patterns and the Methylatoin cellular characteristics.
Progress in the development of pharmacologic inhibitors differs widely between individual enzyme families. The development of histone methyltransferase inhibitors is still in an early preclinical stage.
Several histone deacetylase inhibitors are currently being tested in phase I and phase II clinical trials. However, many other cellular proteins are acetylated, including key regulators of tumor cell growth, and so it is unclear whether the growth inhibition induced by histone deacetylase inhibitors is the result of alterations in the histone acetylation patterns or alterations in signaling pathways that regulate cell proliferation.
DNA methyltransferase inhibitors are at a more clinically advanced stage of development than inhibitors of histone deacetylases or histone methyltransferases, having been extensively tested in phase I—III clinical trials.
In addition, the prototypical DNA methyltransferase inhibitor 5-azacytidine i. DNA methyltransferase activity has an important role in tumor growth, and the activity of DNA methyltransferase inhibitors can be analyzed directly at the DNA level.
Consequently, in this review, we will focus on the role of DNA methyltransferase inhibitors in the further development of epigenetic cancer therapies.
We also discuss future areas of research, including the development of nonnucleoside inhibitors, the application of novel bioanalytical tools for DNA methylation analysis, and the need to optimize treatment schedules on the basis of defined molecular endpoints that allow changes induced by demethylating drugs in patients to be monitored.
The archetypal DNA methyltransferase inhibitor 5-azacytidine, a simple derivative of the nucleoside cytidine Fig. Its demethylating activity was discovered later as the result of its ability to influence cellular differentiation DNA methyltransferases methylate both cytosine residues and 5-azacytosine residues in the DNA.
However, 5-azacytosine prevents the resolution of a covalent reaction intermediate 14which leads to DNA methyltransferase being trapped and inactivated in the form of a covalent protein—DNA adduct Fig.
As a result, cellular DNA methyltransferase is rapidly depleted, and concomitantly genomic DNA is demethylated as a result of continued DNA replication. However, before all 5-azacytidine is converted to a deoxyribonucleoside triphosphate, a portion of it is incorporated into RNA, which affects a variety of RNA functions including ribosome biogenesis 15 and, therefore, has cellular consequences independent of demethylation DNA methyltransferase DNMT inhibitors and their inhibitory mechanisms.
The nucleoside inhibitors 5-azacytidine, 5-azadeoxycytidine, and zebularine are extensively metabolized by cellular pathways small arrows before being incorporated into DNA.
After incorporation, they function as suicide substrates for DNMT enzymes. The nonnucleoside inhibitors procaine, epigallocatechingallate EGCGand RG have been proposed to inhibit DNA methyltransferases by masking DNMT target sequences i.
DNA methyltransferase DNMT inhibition by enzyme trapping or enzyme blocking. Left panel Aza-nucleotides can become incorporated into DNA during replication and then are recognized by DNMT enzymes. A stable reaction intermediate is formed via the sulfhydryl side chain of the catalytic cysteine residue.
Thus, DNMT is trapped and concomitantly degraded. By this mechanism, cells are depleted of DNMT protein. Right panel Small molecules, such as RG, can block the catalytic pocket of free DNMT proteins without the formation of covalent reaction intermediates.
This compound does not need to be modified to a deoxy form and can be more directly incorporated into DNA Fig. Therefore, decitabine may be more specific and less toxic than 5-azacytidine; indeed, the drug shows greater inhibition of DNA methylation and antitumor activity in experimental models Decitabine has single-agent activity in myeloid malignancies 1718including myelodysplastic syndrome, acute myelogenous leukemia, and chronic myelogenous leukemia for details, see below.
However, decitabine also has substantial toxic effects, in particular myelosuppression with neutropenic fever 19that may be linked to the formation of covalent adducts between DNA and trapped DNA methyltransferase proteins These toxic effects highlight one of the central problems in interpreting much of the laboratory and clinical data for DNA methyltransferase inhibitors: It is often not clear whether effects on gene expression and cellular phenotype or even on antitumor activity associated with inhibitor treatment are due to cytotoxicity or to the demethylation of genomic DNA.
Indeed, a recent phase II study of decitabine in patients with chronic myelogenous leukemia appeared to indicate that drug-induced hypomethylation of DNA in peripheral blood cells was less pronounced in responders than in nonresponders The most recent addition to the group of DNA methyltransferase inhibitors is zebularine 21another derivative of 5-azacytidine Fig.
After several chemical modifications, zebularine is incorporated into DNA as a cytosine analogue. Zebularine is more stable than 5-azacytidine or decitabine and may also be less toxic. Orally ingested zebularine causes detectable demethylation and inhibits tumor growth in nude mice However, the oral bioavailability of zebularine in monkeys appears to be low 23and the drug has yet to be evaluated in clinical trials.
Although zebularine appears to have some specificity toward cancer cells 24its mechanism of action is similar to that of the aza-nucleoside inhibitors. Thus, the demethylating activity of zebularine may also be difficult to separate from the toxic effects of DNA methyltransferase depletion that result from covalent enzyme trapping.
In fact, the inherent cytotoxicity of nucleoside DNA methyltransferase inhibitors poses a considerable limitation for their further development as therapeutic agents.
However, their effectiveness in reversing epimutations warrants further investigations to optimize clinical treatment schedules, so that the risks associated with these drugs might be reduced and the benefits maximized. Some nonnucleoside compounds can also inhibit DNA methyltransferase activity.
These substances directly block DNA methyltransferase activity and therefore do not appear to have the inherent toxicity caused by the covalent trapping of the enzyme Fig. One nonnucleoside DNA methyltransferase inhibitor is — -epigallocatechingallate EGCGthe main polyphenol compound in green tea.
EGCG affects various biologic pathways 25 and inhibits DNA methyltransferase activity in protein extracts and in human cancer cell lines After examining the chemical structure of EGCG, Fang et al. However, degradation of EGCG generates a substantial amount of the strong oxidizing agent hydrogen peroxide 27and the oxidation of DNA methyltransferases and other proteins might contribute to the inhibition of DNA methylation by EGCG in vitro and to its cytotoxicity in human cell lines.
Oxidation of DNA methyltransferases may also be involved in the mechanism of action of organoselenium compounds, such as benzyl selenocyanate 28 ; however, to our knowledge, no organoselenium compound has been shown to inhibit DNA methylation under in vivo conditions.
The discovery of most DNA methyltransferase inhibitors discussed above was fortuitous. The rational design of DNA methyltransferase inhibitors has been hampered by the lack of three-dimensional structures for the most relevant eukaryotic DNA methyltransferases, and it has been difficult to establish stringent DNA methyltransferase assays that are suitable for high-throughput screening.
However, a three-dimensional homology model for the human DNA methyltransferase1 catalytic domain has been established 29 and used in an in silico screening assay to identify RG, a small-molecule inhibitor of human DNA methyltransferases RG appears to block the active site of DNA methyltransferase Fig.
The inhibitory mechanism of RG also appears to be direct and specific for DNA methyltransferases, in that RG has comparatively low toxicity in human cancer cell lines Thus, RG is an attractive candidate for further evaluation as a lead compound for new drug development.
The group of nonnucleoside DNA methyltransferase inhibitors also contains three additional classes of less well-characterized compounds. Procaine appears to bind to CpG-rich sequences and thereby block the binding of DNA methyltransferases to DNA Fig.
However, procaine must be present in a high concentration — μ M to be an effective DNA methyltransferase inhibitor, and it has not been effective in all cell lines tested The psammaplins also inhibit histone deacetylase activity 34 and thus should be evaluated further as inhibitors of both histone deacetylases and DNA methyltransferases.
Hairpin loops have been used as competitor substrates for DNA methyltransferases in mouse erythroleukemia cells and have been able to induce the weak expression of the p16 tumor suppressor gene in human HT29 colon carcinoma cells In addition, transfection of human HCT and SW48 colon cancer cell lines with antisense oligonucleotides against DNA methyltransferase 1 resulted in the demethylation and reactivation of p16 Because only a limited response was observed in trials of single-agent MG98 therapy, current phase II trials in metastatic renal cell cancer are evaluating combination therapy of MG98 and interferon.
Alterations in the level or pattern of genomic DNA methylation may be an important endpoint for analyzing the effect of DNA methyltransferase inhibitor treatment.
Changes in DNA methylation patterns can be analyzed with various methods, but most methods have substantial limitations, as outlined in Table 1. Chromatographic and electrophoretic analysis of genomic DNA, for instance, allows for a straightforward determination of the methylation level of the whole genome but does not provide information about the distribution of methylated bases, i.
In contrast, bisulfite sequencing provides a detailed map of DNA methylation patterns, but the method is very time-consuming and limited to a few hundred base pairs of DNA per experiment.
In addition, microarray analysis may allow a genome-wide analysis of DNA methylation patterns at a high resolution, but this technology is still challenging experimentally, and better microarray platforms need to be developed for such analyses.
: Methylation inhibitors for cancer prevention| Frontiers | DNA Methyltransferases: A Novel Target for Prevention and Therapy | Aging Methypation— Acne solutions High-potency natural fat burner, Canceg Y, Prevenhion C et al Ihibitors of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. MicroRNA expression profiles classify human cancers. Science —9. Lander ES, Linton LM, Birren B, et al. In contrast, the reactivation of expression of p16 INK4A and p15 INK4B was implicated in the reduced cytotoxicity and growth inhibition and a lack of DNA damage effects when DNMT1 gene was deleted or disrupted. |
| Introduction | For example, in vitro studies have shown that decitabine reverses resistance to various anticancer agents in vitro 41 , and a clinical trial of this approach in ovarian cancer is ongoing. One nonnucleoside DNA methyltransferase inhibitor is — -epigallocatechingallate EGCG , the main polyphenol compound in green tea. The areas of cells were selected based on DAPI staining, excluding thick cellular areas with overlapping cells, areas with artifacts, or poor staining. Article PubMed PubMed Central Google Scholar Liu K, Dong F, Gao H, et al. Article CAS PubMed Central Google Scholar Santi DV, Norment A, Garrett CE. Leukemia 12 3 —6. Consequently, the epigenetic mechanism through DNA methylation facilitates the organization of the genome into active euchromatin and inactive regions heterochromatin with respect to gene transcription. |
| Frontiers | DNMT Inhibitors Increase Methylation in the Cancer Genome | Cancer Sci — Proc Prevebtion Acad Sci USA 38 —5. Heery, D. Inhibiyors, Methylation inhibitors for cancer prevention. The same efforts have been made to develop inhibitors for sirtuins, the class III HDACs. As a result, cellular DNA methyltransferase is rapidly depleted, and concomitantly genomic DNA is demethylated as a result of continued DNA replication. |
| Top bar navigation | Identification of hypermethylated genes associated with cisplatin preventiin in human cancers. Cell Enzymes for enzyme deficiency. These data provide further Preventiob evidence to demonstrate Vibrant epigenetic alterations may contribute to the initiation and promotion of liver carcinogenesis. Chemotherapy has limited applicability in tumor therapy because of the associated complications, including nausea, vomiting, myelosuppression and resistance. The interconnection between DNA methylation and the demethylation processes is exemplified in Fig. |
Video
Epigenetic therapy: a new frontier for cancer treatment - Dr Clare Stirzaker
Es ist die richtigen Informationen