Enhancement of the Antileukemic Action of the Inhibitors of DNA and Histone Methylation: 5-Aza-2'-Deoxycytidine and 3-Deazaneplanocin-A by Vitamin C.

Epigenetic gene silencing by DNA methylation and histone methylation by EZH2 play an important role in the development of acute myeloid leukemia (AML). EZH2 catalyzes the trimethylation of histone H3-lysine 27-trimethylated (H3K27me3). These epigenetic alterations silence the expression of the genes that suppress leukemogenesis. Reversal of this gene silencing by 5-aza-2'-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, and by 3-deazaneplanocin-A (DZNep), an inhibitor of EZH2, results in synergistic gene reactivation and antileukemic interaction. The objective of this study is to determine if the addition of another epigenetic agent could further enhance the antileukemic action of these inhibitors of DNA and histone methylation. Vitamin C (Vit C) is reported to enhance the antineoplastic action of 5-Aza-CdR on AML cells. The mechanism responsible for this action of Vit C is due to its function as a cofactor of alpha-ketoglutarate-dependent dioxygenases (α-KGDD). The enhancement by Vit C of the catalytic activity of α-KGDD of the ten eleven translocation (TET) pathway, as well as of the Jumonji C histone demethylases (JHDMs), is shown to result in demethylation of DNA and histones, leading to reactivation of tumor suppressor genes and an antineoplastic effect. This action of Vit C has the potential to complement the antileukemic action of 5-Aza-CdR and DZNep. We observe that Vit C remarkably increases the antineoplastic activity of 5-Aza-CdR and DZNep against myeloid leukemic cells. An important step to bring this novel epigenetic therapy to clinical trial in patients with AML is the determination of its optimal dose schedule.

Epigenetic Modulation of Self-Renewal Capacity of Leukemic Stem Cells and Implications for Chemotherapy.

Most patients with acute myeloid leukemia (AML) have a poor prognosis. Curative therapy of AML requires the complete eradication of the leukemic stem cells (LSCs). One aspect of LSCs that is poorly understood is their low frequency in the total population of leukemic cells in AML patients. After each cell division of LSCs, most of the daughter cells lose their capacity for self-renewal. Investigations into the role of Isocitrate dehydrogenase (IDH) mutations in AML provide some insight on the regulation of the proliferation of LSCs. The primary role of IDH is to convert isocitrate to alpha-keto-glutarate (α-KG). When IDH is mutated, it converts α-KG to 2-hydroxyglutarate (2-HG), an inhibitor of the TET pathway and Jumonji-C histone demethylases (JHDMs). The demethylating action of these enzymes removes the epigenetic gene-silencing markers, DNA methylation, H3K27me3 and H3K9me2 and can lead to the differentiation of LSCs. This enzymatic action is blocked by 2-HG in mutated IDH (mut-IDH) AML patients, who can be induced into remission with antagonists of 2-HG. These observations suggest that there exists in cells a natural enzymatic mechanism that uses demethylation to reverse epigenetic gene-silencing, leading to a loss of the self-renewal capacity of LSCs. This mechanism limits the proliferative potential of LSCs. Epigenetic agents that inhibit DNA and histone methylation exhibit a synergistic antineoplastic action on AML cells. It is possible that the therapeutic potential of this epigenetic therapy may be enhanced by demethylation enzymes, resulting in a very effective treatment for AML.

Inhibition of DNA and Histone Methylation by 5-Aza-2'-Deoxycytidine (Decitabine) and 3-Deazaneplanocin-A on Antineoplastic Action and Gene Expression in Myeloid Leukemic Cells.

Epigenetic alterations play an important role in the development of acute myeloid leukemia (AML) by silencing of genes that suppress leukemogenesis and differentiation. One of the key epigenetic changes in AML is gene silencing by DNA methylation. The importance of this alteration is illustrated by the induction of remissions in AML by 5-aza-2'-deoxycytidine (5-AZA-CdR, decitabine), a potent inhibitor of DNA methylation. However, most patients induced into remission by 5-AZA-CdR will relapse, suggesting that a second agent should be sought to increase the efficacy of this epigenetic therapy. An interesting candidate for this purpose is 3-deazaneplanocin A (DZNep). This analog inhibits EZH2, a histone methyltransferase that trimethylates lysine 27 histone H3 (H3K27me3), a marker for gene silencing. This second epigenetic silencing mechanism also plays an important role in leukemogenesis as shown in preclinical studies where DZNep exhibits potent inhibition of colony formation by AML cells. We reported previously that 5-AZA-CdR in combination with DZNep exhibits a synergistic antineoplastic action against human HL-60 AML cells and the synergistic activation of several tumor suppressor genes. In this report, we showed that this combination also induced a synergistic activation of apoptosis in HL-60 cells. The synergistic antineoplastic action of 5-AZA-CdR plus DZNep was also observed on a second human myeloid leukemia cell line, AML-3. In addition, 5-AZA-CdR in combination with the specific inhibitors of EZH2, GSK-126, or GSK-343, also exhibited a synergistic antineoplastic action on both HL-60 and AML-3. The combined action of 5-AZA-CdR and DZNep on global gene expression in HL-60 cells was investigated in greater depth using RNA sequencing analysis. We observed that this combination of epigenetic agents exhibited a synergistic activation of hundreds of genes. The synergistic activation of so many genes that suppress malignancy by 5-AZA-CdR plus DZNep suggests that epigenetic gene silencing by DNA and histone methylation plays a major role in leukemogenesis. Targeting DNA and histone methylation is a promising approach that merits clinical investigation for the treatment of AML.

Targeting of cancer stem cells by inhibitors of DNA and histone methylation.

INTRODUCTION: Curative chemotherapy should target cancer stem cells (CSCs). The key characteristics of CSCs are a block in differentiation and an epigenetic signature similar to embryonic stem cells (ESCs). Differentiation by ESCs and CSCs is suppressed by gene silencing through the polycomb repressive complex 2 (PRC2) and/or DNA methylation. PRC2 contains the EZH2 subunit, which catalyzes the trimethylation of histone 3 lysine 27, a gene silencing marker. It is possible to reverse this 'double lock' mechanism using a combination of inhibitors of EZH2 and DNA methylation (5-aza-2'-deoxycytidine), which exhibits remarkable synergistic antineoplastic activity in preclinical studies. AREAS COVERED: The authors discuss several specific EZH2 inhibitors that have been synthesized with antineoplastic activity. One such inhibitor, EPZ-6438 (E7438), has been shown to be effective against lymphoma in a Phase I study. The indirect EZH2 inhibitor, 3-deazaneplanocin-A (DZNep), also exhibits remarkable anticancer activity due to its inhibition of methionine metabolism. EXPERT OPINION: Agents that target EZH2 warrant Phase I trials. Due to its positive pharmacodynamics, DZNep merits a high priority for clinical investigation. Agents that show positive results in Phase I studies should be advanced to clinical trials for use in combination with 5-aza-2'-deoxycytidine due to the interesting potential of this epigenetic therapy to target CSCs.

Epigenetic therapy of acute myeloid leukemia using 5-aza-2'-deoxycytidine (decitabine) in combination with inhibitors of histone methylation and deacetylation.

BACKGROUND: The silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation occurs frequently in acute myeloid leukemia (AML). This epigenetic alteration can be reversed by 5-aza-2'-deoxcytidine (decitabine, 5-AZA-CdR). Although 5-AZA-CdR can induce complete remissions in patients with AML, most patients relapse. The effectiveness of this therapy may be limited by the inability of 5-AZA-CdR to reactivate all TSGs due to their silencing by other epigenetic mechanisms such as histone methylation or chromatin compaction. EZH2, a subunit of the polycomb repressive complex 2, catalyzes the methylation of histone H3 lysine 27 (H3K27) to H3K27me3. 3-Deazaneplanocin-A (DZNep), an inhibitor of methionine metabolism, can reactivate genes silenced by H3K27me3 by its inhibition of EZH2. In a previous report, we observed that 5-AZA-CdR, in combination with DZNep, shows synergistic antineoplastic action against AML cells. Gene silencing due to chromatin compaction is attributable to the action of histone deacetylases (HDAC). This mechanism of epigenetic gene silencing can be reversed by HDAC inhibitors such as trichostatin-A (TSA). Silent TSGs that cannot be reactivated by 5-AZA-CdR or DZNep have the potential to be reactivated by TSA. This provides a rationale for the use of HDAC inhibitors in combination with 5-AZA-CdR and DZNep to treat AML. RESULTS: The triple combination of 5-AZA-CdR, DZNep, and TSA induced a remarkable synergistic antineoplastic effect against human AML cells as demonstrated by an in vitro colony assay. This triple combination also showed a potent synergistic activation of several key TSGs as determined by real-time PCR. The triple combination was more effective than the combination of two agents or a single agent. Microarray analysis showed that the triple combination generated remarkable changes in global gene expression. CONCLUSIONS: Our data suggest that it may be possible to design a very effective therapy for AML using agents that target the reversal of the following three epigenetic "lock" mechanisms that silence gene expression: DNA methylation, histone methylation, and histone deacetylation. This approach merits serious consideration for clinical investigation in patients with advanced AML.

Optimization of cytarabine (ARA-C) therapy for acute myeloid leukemia.

Cytarabine (cytosine arabinoside) is one of the most effective drugs for the treatment of acute myeloid leukemia. The standard dose of cytarabine used to treat this leukemia is 100 mg per square meter. In an attempt to improve the effectiveness of cytarabine against acute myeloid leukemia, a high-dose treatment (3,000 mg per square meter) was introduced into therapy. The side effects of high-dose cytarabine was a major concern, especially its neurological toxicity. A review of recent clinical trials indicates that this high-dose cytarabine can be replaced by the intermediate-dose of 1,000 mg per square meter without loss of efficacy and with less toxicity. This is an important step to improve the efficacy of cytarabine for the treatment of acute myeloid leukemia. Despite the improvements in the therapy for this leukemia, the current overall survival rate for adult patients is less than 30%. To optimize the cytarabine therapy, it is important to determine how some leukemic stem cells survive treatment. Preclinical data suggest that survival of the leukemic stem cells could be due to the long 12 hour interval between infusions of cytarabine, which permits some leukemic cells to escape its S phase specific action. Among the other factors that can lead to leukemic cell survival are the high levels in the liver and spleen of cytidine deaminase, the enzyme that inactivates cytarabine and drug resistance due to deficiency in deoxycytidine kinase, the enzyme that activates the prodrug, cytarabine. Several approaches are proposed in this commentary to overcome these impediments with the goal of increasing the effectiveness of cytarabine for the treatment of acute myeloid leukemia.

Epigenetic therapy of non-small cell lung cancer using decitabine (5-aza-2'-deoxycytidine).

Epigenetic analysis shows that many genes that suppress malignancy are silenced by aberrant DNA methylation in lung cancer. Many of these genes are interesting targets for reactivation by the inhibitor of DNA methylation, decitabine (5-aza-2'-deoxycytidine, DAC). A pilot study on intense dose DAC showed promising results in patients with metastatic non-small cell lung cancer (NSCLC). However, subsequent clinical studies using low dose DAC were not very effective against NSCLC and interest in this therapy diminished. Recently, interesting responses were observed in a patient with NSCLC following treatment with a combination of the related inhibitor of DNA methylation, 5-azacytidine, and an inhibitor of histone deacetylation. This finding has generated a renewed interest in the epigenetic therapy of lung cancer. Preclinical studies indicate that DAC has remarkable chemotherapeutic potential for tumor therapy. This epigenetic agent has a delayed and prolonged epigenetic action on tumor cells. This delayed action should be taken into consideration in the design and evaluation of clinical studies on DAC. Future research should be directed at finding the optimal dose-schedule of de DAC for the treatment of NSCLC.

Epigenetic action of decitabine (5-aza-2'-deoxycytidine) is more effective against acute myeloid leukemia than cytotoxic action of cytarabine (ARA-C).

Treatment of elderly patients with acute myeloid leukemia (AML) with standard cytarabine (ARA-C) chemotherapy can achieve some complete responses (CR), but the median overall survival is less than one year. New approaches should be investigated. The inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (decitabine, DAC), shows effectiveness in these patients, but was not approved by the US Federal Drug Administration. This decision was based on a clinical trial where DAC showed a median survival of 7.0 months as compared to standard ARA-C therapy or supportive care of 5.0 months. However, the difference was not statistically significant. Preclinical data indicate that DAC is much more effective against human AML than ARA-C. The key question is should these preclinical data also be used in the evaluation of new drugs for the clinical treatment of AML? The delayed epigenetic action of DAC is very different than the acute cytotoxic action of ARA-C and should be taken into account in the design clinical trials and evaluation of the response.

Pharmacokinetic and pharmacodynamic analysis of 5-aza-2'-deoxycytidine (decitabine) in the design of its dose-schedule for cancer therapy.

5-Aza-2'-deoxycytidine (5-AZA-CdR, decitabine), an epigenetic drug that inhibits DNA methylation, is currently used to treat myelodysplastic syndrome (MDS), and is under investigation for treating acute myeloid leukemia (AML) and other malignancies. 5-AZA-CdR can reactivate tumor suppressor genes silenced by aberrant DNA methylation, a frequent event in all types of cancer. Because this epigenetic change is reversible, it is a good target for 5-AZA-CdR therapy. We have reviewed the preclinical data of 5-AZA-CdR to analyze the concentrations and exposure times required to eradicate cancer stem cells. We analyzed the dose-schedules used in animal models that show potent antineoplastic activity of 5-AZA-CdR. We attempted to correlate the preclinical data with the responses obtained in clinical trials of 5-AZA-CdR in patients with cancer. The pharmacokinetics and drug distribution of 5-AZA-CdR are key parameters because adequate therapeutic drug levels are required to eliminate cancer stem cells in all anatomic compartments. The plasma half-life of 5-AZA-CdR in humans is approximately 20 minutes due to the high levels in the liver of cytidine deaminase, the enzyme that inactivates this analogue. This provides a rationale to use an inhibitor of cytidine deaminase in combination with 5-AZA-CdR. Low-dose 5-AZA-CdR is effective for MDS and AML and can induce complete remissions (CR). However, maintenance of CR with low-dose 5-AZA-CdR is difficult. Based on analyses of preclinical and clinical data, low dose 5-AZA-CdR has the potential to be an effective form of therapy in some patients with cancer. For patients who do not respond to low dose therapy we recommend dose-intensive treatment with 5-AZA-CdR. Patients who are candidates for intensive dose 5-AZA-CdR should have a good bone marrow status so as to permit adequate recovery from myelosuppression, the major toxicity of 5-AZA-CdR. Solid tumors are also interesting targets for therapy with 5-AZA-CdR. Both low dose and intensive therapy with 5-AZA-CdR can reduce the proliferative potential of tumor stem cells in animal models. We propose novel dose schedules of 5-AZA-CdR for investigation in patients with cancer. The full chemotherapeutic potential of 5-AZA-CdR to treat cancer merits further clinical investigation and can only be realized when its optimal dose-schedule is determined.

Synergistic antileukemic action of a combination of inhibitors of DNA methylation and histone methylation.

DNA methylation and histone methylation are both involved in epigenetic regulation of gene expression and their dysregulation can play an important role in leukemogenesis. Aberrant DNA methylation has been reported to silence the expression of tumor suppressor genes in leukemia. Overexpression of the histone methyltransferase, EZH2, a subunit of the polycomb group repressive complex 2 (PRC2), was observed to promote oncogenesis. This is due to aberrant gene silencing by the trimethylation of histone H3 lysine 27 (H3K27me3) by EZH2. Since both these epigenetic silencing events are reversible, they are interesting targets for chemotherapeutic intervention by using an inhibitor of DNA methylation, such as 5-aza-2'-deoxcytidine (5-AZA-CdR), and 3-deazaneplanocin-A (DZNep), an inhibitor of the EZH2. Human HL-60 and murine L1210 leukemic cells exposed in vitro to 5-AZA-CdR and DZNep in combination showed a synergistic loss of clonogenicity in a colony assay as compared to each agent alone. This positive chemotherapeutic interaction was also observed in mice with L1210 leukemia. Quantitative PCR showed that the combination also produced a remarkable synergistic activation of the tumor suppressor genes, CDKN1A and FBXO32. Microarray analysis showed that 5-AZA-CdR plus DZNep produced a synergistic activation of >150 genes. Our results indicate that 5-AZA-CdR plus DZNep can reactivate target genes that are silenced by two distinct epigenetic mechanisms leading to a loss of the proliferative potential of leukemic cells.

A Perspective on the Comparative Antileukemic Activity of 5-Aza-2'-deoxycytidine (Decitabine) and 5-Azacytidine (Vidaza).

5-Aza-2'-deoxycytidine (5-AZA-CdR, decitabine, Dacogen®) and 5-azacytidine (5-AC, Vidaza®) are epigenetic agents that have been approved for the clinical treatment of the hematological malignancy myelodysplastic syndrome (MDS) and are currently under clinical evaluation for the treatment of acute myeloid leukemia (AML). Most investigators currently classify 5-AZA-CdR and 5-AC as inhibitors of DNA methylation, which can reactivate tumor suppressor genes silenced by this epigenetic event. Examination of the pharmacology of these analogues reveals important differences with respect to their molecular mechanism of action. The action of 5-AZA-CdR is due to its incorporation into DNA. 5-AC is a riboside analogue that is incorporated primarily into RNA. A small fraction of 5-AC is converted to its deoxyribose form by ribonucleotide reductase and subsequently incorporated into DNA. The incorporation of 5-AC into RNA can interfere with the biological function of RNA and result in an inhibition protein synthesis. Microarray analysis revealed that both these analogues target the expression of different cohorts of genes. Preclinical studies show that 5-AZA-CdR is a more effective antileukemic agent than 5-AC. One explanation for this observation is that 5-AC blocks the progression of some leukemic cells from G1 into S phase, and this protects these cells from the chemotherapeutic action of this riboside analogue related to its incorporation into DNA. However, differences in chemotherapeutic efficacy of these related analogues have not been clearly demonstrated in clinical trials in patients with hematological malignancies. These observations should be taken into consideration in the design of new clinical trials using 5-AZA-CdR or 5-AC in patients with MDS and AML.

3-Deazauridine enhances the antileukemic action of 5-aza-2'-deoxycytidine and targets drug-resistance due to deficiency in deoxycytidine kinase.

New approaches should be sought to treat high-risk acute lymphoblastic leukemia (ALL). Since aberrant DNA methylation plays an important role in leukemogenesis of ALL, it can be targeted by 5-aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation. 5-AZA-CdR is a prodrug that is activated by deoxycytidine kinase (DCK). Leukemic cells lacking DCK are drug-resistant. In a previous phase I study, we reported that 5-AZA-CdR could induce remissions in ALL. However, some patients developed drug-resistance due to deficiency in DCK. These observations aroused our interest in 3-deazauridine (3-DU), a CTP synthetase inhibitor that is effective against leukemic cells deficient in DCK. In this report, we observed that 3-DU enhanced the in vitro antineoplastic action of 5-AZA-CdR on human leukemic cells by increasing its incorporation into DNA. Using an optimized dose-schedule we showed that this combination could cure some mice bearing L1210 leukemia, even in the presence of a subpopulation of drug-resistant (L1210/ARA-C) leukemic cells lacking DCK. 3-DU alone also cured some mice with L1210/ARA-C leukemia. In a pilot study on 3 relapsed patients with advanced ALL, the combination of 5-AZA-CdR and 3-DU produced a marked reduction in leukemic blasts, confirming our preclinical observations. Furthermore, after several treatments with these agents all three patients developed drug-resistance to 5-AZA-CdR as determined by an in vitro drug sensitivity test. In two patients we showed by enzymatic analysis that the drug-resistance was due to deficiency in DCK. Our preclinical and clinical results provide a strong rationale to further investigate the combination of 5-AZA-CdR and 3-DU for the treatment of advanced ALL.

Inhibition of cytidine deaminase by zebularine enhances the antineoplastic action of 5-aza-2'-deoxycytidine.

Cytidine (CR) deaminase is a key enzyme in the catabolism of cytosine nucleoside analogues, since their deamination results in a loss of their pharmacological activity. In this report we have investigated the importance of CR deaminase with respect to the antineoplastic action of inhibitors of DNA methylation, 5-aza-2'-deoxycytidine (5-AZA-CdR) and zebularine. Zebularine has a dual mechanism of action, since it can also inhibit CR deaminase. The objective of our study was to investigate the importance of zebularine as an inhibitor of CR deaminase with respect to the antineoplastic action of 5-AZA-CdR. Using an in vitro clonogenic assay, we investigated the antineoplastic action of 5-AZA-CdR and zebularine, alone and in combination on wild type 3T3 murine fibroblasts and corresponding V5 cells transduced with CR deaminase gene to express a very high level of CR deaminase activity. The V5 cells were much less sensitive to 5-AZA-CdR than the wild type 3T3 cells. The addition of zebularine significantly enhanced the antineoplastic action of 5-AZA-CdR on V5 cells, but not 3T3 cells. Enzymatic analysis on CR deaminase purified from the V5 cells showed that zebularine is a competitive inhibitor of the deamination of 5-AZA-CdR. These in vitro observations are in accord with our in vivo study in mice with L1210 leukemia, which showed that zebularine increased the antileukemic activity of 5-AZA-CdR. Pharmacokinetic analysis also showed that zebularine increased the plasma level of 5-AZA-CdR during an i.v. infusion in mice. Our results indicate that the major mechanism by which zebularine enhances the antineoplastic action of 5-AZA-CdR is by inhibition of CR deaminase. These findings provide a rationale to investigate 5-AZA-CdR in combination with zebularine in patients with advanced leukemia.

Synergistic effect of 5-Aza-2'-deoxycytidine and genistein in combination against leukemia.

5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation, is an effective agent for the treatment of leukemia. The aim of this study was to investigate the antileukemic activity of this epigenetic agent in combination with genistein, a nontoxic isoflavone with chemopreventive activity. The combined treatment produced a synergistic loss of clonogenicity in human myeloid (HL-60) and lymphoid (MOLT-3) leukemic cell lines. Genistein alone showed a significant antileukemic activity against murine 5-AZA-CdR-resistant cells, and this effect was enhanced when used in combination with 5-AZA-CdR. The combined treatment also produced a synergistic increase in life span of mice with L1210 leukemia. These results suggest that genistein may have the potential to increase the clinical efficacy of 5-AZA-CdR for the treatment of leukemia.

Preclinical evaluation of the antineoplastic action of 5-aza-2'-deoxycytidine and different histone deacetylase inhibitors on human Ewing's sarcoma cells.

BACKGROUND: Most patients with advanced Ewing's sarcoma (EWS) respond poorly to conventional chemotherapy, indicating the need for new treatment approaches. Epigenetic events, such as promoter hypermethylation and chromatin histone deacetylation, silence the expression of tumor suppressor genes (TSGs) and play an important role in tumorigenesis. These epigenetic changes can be reversed by using 5-aza-2'-deoxycytidine (5AZA-CdR), a potent inhibitor of DNA methylation, in combination with an inhibitor of histone deacetylase (HDAC). RESULTS: Here, we used a clonogenic assay to evaluate the in vitro antineoplastic activity of 5AZA-CdR in combination with different HDAC inhibitors on EWS cells. We observed that the HDAC inhibitors, MS-275, trichostatin-A, phenylbutyrate, LAQ824 and depsipeptide, enhanced the antineoplastic action of 5AZA-CdR on EWS cells. The combination of 5AZA-CdR and MS-275 showed marked synergy, and was correlated with significant reactivation of the expression of two TSGs, E-cadherin and tumor suppressor lung cancer-1 (TSLC1), in a EWS cell line. CONCLUSION: These results suggest the value of future clinical studies investigating the combination of 5AZA-CdR and MS-275 in patients with advanced EWS.

Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer.

BACKGROUND: The inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is a potential target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established. METHODS: Clonogenic assays were performed on leukemic and tumor cell lines to evaluate the in vitro antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by reverse transcriptase-PCR and Line-1 assays. The in vivo antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time, and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy for an in vitro clonogenic assay. RESULTS: Increasing the DAC concentration and duration of exposure produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs (p57KIP2 in HL-60 leukemic cells and p16CDKN2A in Calu-6 lung carcinoma cells) and the inhibition of global DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (> 1 microM). CONCLUSION: We have shown that intensification of the DAC dose markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce in vitro clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is related to its epigenetic action. Our observations provide a strong rationale to perform clinical trials using dose intensification of DAC to maximize the chemotherapeutic potential of this epigenetic agent in patients with cancer.

Antileukemic activity of genistein, a major isoflavone present in soy products.

Soy has been used in traditional medicine for the treatment of various diseases, including cancer. The isoflavones present in soy have been shown in animal models to have cancer-preventing activity. However, the therapeutic effects of isoflavones against cancer are still unclear. We have evaluated the in vitro and in vivo antileukemic activity of genistein (1), a major isoflavone present in soy. We observed that it produced a dose- and time-dependent antineoplastic activity against myeloid and lymphoid leukemic cell lines. In addition, genistein treatment of the leukemic cells reactivated tumor suppressor genes that were silenced by aberrant DNA methylation. A genistein-enriched diet produced a moderate, but significant, antileukemic effect in mice. The limited extent of this in vivo response may have been due to the rapid metabolic inactivation of genistein in mice. Due to the longer half-life of genistein in humans, a soy-enriched diet has the potential to produce plasma levels of this isoflavone in the range of the concentrations used in vitro that produced an antileukemic activity.

Effect of histone deacetylase inhibitor LAQ824 on antineoplastic action of 5-Aza-2'-deoxycytidine (decitabine) on human breast carcinoma cells.

PURPOSE: Epigenetic silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation and chromatin deacetylation provides interesting targets for chemotherapeutic intervention by inhibitors of these events. 5-Aza-2'-deoxycytidine (decitabine, 5AZA-CdR) is a potent demethylating agent, which can reactivate TSGs silenced by aberrant DNA methylation. LAQ824 (LAQ) is a novel inhibitor of histone deacetylase (HDAC) that shows antineoplastic activity and can activate genes that produce cell cycle arrest. Both 5AZA-CdR and LAQ as single agents are currently under clinical investigation in patients with cancer. Previous reports indicate that the "cross-talk" between inhibitors of DNA methylation and HDAC can result in a synergistic activation of silent TSGs. These observations suggest that combination of these inhibitors may be an effective form of epigenetic therapy for breast cancer. The objective of our study was to determine if the combination of 5AZA-CdR and LAQ would show additive or synergistic antineoplastic activity on human MDA-MB-231 and MCF-7 breast carcinoma cells. The antineoplastic activity of these agents was evaluated by clonogenic assay and inhibition of DNA synthesis. RESULTS: The combination produced greater antineoplastic activity for the MDA-MB-231 tumor cells than either agent alone. For the MCF-7 tumor cells, there were signs of antagonism between 5AZA-CdR and LAQ when administered simultaneously. When a sequential schedule (first 5AZA-CdR followed by LAQ) was used, there were no signs of antagonism of the antineoplastic action for the MCF-7 tumor cells. The mechanism of this interaction is probably due to the reduction of progression of MCF-7 tumor cells into S phase by LAQ. This would interfere with the antineoplastic action of 5AZA-CdR, since it is an S phase specific agent. CONCLUSIONS: These studies demonstrated the importance of the schedule of administration of 5AZA-CdR and LAQ and may have application for future clinical trials on the treatment of breast cancer with these agents.

Epigenetic therapy of cancer with 5-aza-2'-deoxycytidine (decitabine).

Epigenetic events, such as aberrant DNA methylation, have been demonstrated to silence the expression of many genes that suppress malignancy. Since the event is reversible, it is an interesting target for intervention with specific inhibitors of DNA methylation, such as 5-aza-2'-deoxycytidine (5-AZA-CdR, decitabine). 5-AZA-CdR is a prodrug that requires activation via phosphorylation by deoxcytidine kinase. The nucleotide analog is incorporated into DNA, where it produces an irreversible inactivation of DNA methyltransferase. 5-AZA-CdR is an S-phase-specific agent. The demethylation of DNA by this analog in neoplastic cells can lead to the reactivation of silent tumor-suppressor genes, induction of differentiation or senescence, growth inhibition, and loss of clonogenicity. 5-AZA-CdR was demonstrated to be a potent antineoplastic agent against leukemia and tumors in animal models. Preliminary clinical trials of 5-AZA-CdR using different dose-schedules have shown interesting antineoplastic activity in patients with leukemia, myelodysplastic syndrome (MDS), and non-small cell lung cancer (NSCLC). Pharmacokinetic studies have shown that 5-AZA-CdR has a short in vivo half-life of 15 to 25 minutes. The major toxicity produced by this analog is granulocytopenia. To exploit the full chemotherapeutic potential of 5-AZA-CdR for the treatment of cancer, its optimal dose-schedule has to be found. This will require a good understanding of the pharmacology of this analog and its action on both normal and neoplastic cells.

Pharmacology of 5-Aza-2'-deoxycytidine (decitabine).

The preclinical pharmacology of 5-aza-2'-deoxycytidine (decitabine, 5AZA-CdR) is reviewed. 5AZA-CdR, an analogue of deoxycytidine, is a prodrug that requires metabolic activation by deoxycytidine kinase. The active inhibitor in the cell is its triphosphate form (5AZA-dCTP), which incorporates very readily into DNA to produce an inhibition of DNA methyltransferase. The mechanism responsible for the antileukemic action of 5AZA-CdR is related to its reversal of epigenetic silencing by aberrant DNA methylation of genes that suppress leukemiogenesis. 5AZA-CdR is an S-phase-specific agent. At concentrations in the range of micromolars this analogue can induce terminal differentiation and loss of clonogenicity of human leukemic cells. Drug resistance to 5AZA-CdR occurs primarily by reduction in deoxycytidine kinase activity or increase in the activity of cytidine deaminase, the enzyme that inactivates this analogue. 5AZA-CdR is a very potent antileukemic agent in animal models, more effective than the related antileukemic drug, cytosine arabinoside. In humans, 5AZA-CdR has a short half-life of 15 to 25 minutes due to rapid inactivation by liver cytidine deaminase. The major toxicity produced by 5AZA-CdR is myelosuppression. Preliminary clinical studies in patients with hematologic malignancies indicate that 5AZA-CdR is an active chemotherapeutic agent. The optimal dose-schedule for this interesting epigenetic agent with a novel mechanism of action remains to be determined. Translation of the pharmacology of 5AZA-CdR into therapeutic regimens based on scientific rationale can be used to obtain this objective.