Methionine restriction of glioma does not induce MGMT and greatly improves temozolomide efficacy in an orthotopic nude-mouse model: A potential curable approach to a clinically-incurable disease.

Glioma is a highly recalcitrant disease with a 5-year survival of 6.8 %. Temozolomide (TMZ), first-line therapy for glioma, is more effective in O6-methylguanine-DNA methyltransferase (MGMT)-negative gliomas than in MGMT-positive gliomas as MGMT confers resistance to TMZ. Methionine restriction is effective for many cancers in mouse models including glioma. The concern is that methionine restriction could induce MGMT by decreasing DNA methylation and confer resistance to TMZ. In the present study, we investigated the efficacy of combining methionine restriction with TMZ for the treatment of MGMT-negative glioma, and whether methionine restriction induced MGMT. Human MGMT-negative U87 glioma cells were used to determine the efficacy of TMZ combined with methionine restriction. Recombinant methioninase (rMETase) inhibited U87 glioma growth without induction of MGMT in vitro. The combination of rMETase and TMZ inhibited U87 cell proliferation more than either agent alone in vitro. In the orthotopic nude-mouse model, the combination of TMZ and a methionine-deficient diet was much more effective than TMZ alone: two mice out of five were cured of glioma by the combination. No mice died during the treatment period. Methionine restriction enhanced the efficacy of TMZ in MGMT-negative glioma without inducing MGMT, demonstrating potential clinical promise for improved outcome of a currently incurable disease.

[Curcumol reverses temozolomide resistance in glioma cells by regulating the UTX/MGMT axis].

OBJECTIVE: To explore the mechanism through which curcumol reverses primary drug resistance in glioma cells. METHODS: The inhibitory effect of 10, 20, and 40 µg/mL curcumol were observed in human glioma cell lines A172 and U251. UTX-overexpressing glioma cells constructed by lentiviral transfection were treated with curcumol (40 µg/mL), temozolomide (TMZ; 10 µg/mL), or both, and the changes in cell viability, clone formation capacity and apoptosis were assessed using MTT assay, cell clone formation experiment, and flow cytometry; UTX activity in the cells was determined using a UTX detection kit, and the enrichment of UTX and H3K27me3 in the MGMT promoter region was detected with ChiP-qPCR. The protein expressions in glioma cells were detected using Western blotting and immunohistochemistry. In a nude mouse model bearing glioma xenografts, the effects of curcumol (20 mg/kg), TMZ (20 mg/kg) and their combination on tumor growth and expressions of UTX, H3K27me3 and MGMT were evaluated. RESULTS: Curcumol significantly inhibited the proliferation (P<0.05) and promoted apoptosis of cultured glioma cells (P<0.01). Curcumol, but not TMZ, produced significant inhibitory effect on tumor growth in the tumor-bearing mice (P<0.01). Curcumol significantly inhibited UTX activity and increased the expression level of H3K27me3 protein in the glioma cells. UTX overexpression obviously decreased H3K27me3 protein expression and reversed the effects of curcumol on glioma cell proliferation and apoptosis (P<0.01). Curcumol reduced the enrichment of UTX and H3K27me3 in the MGMT promoter region (P<0.05) and decreased MGMT protein expression, which was reversed by UTX overexpression. In both the in vivo and in vitro experiments, curcumol combined with TMZ significantly increased H3K27me3 protein expression in the glioma cells, reduced the expression of its downstream target gene MGMT, and enhanced TMZ sensitivity of the glioma cells. CONCLUSION: Curcumol can enhance glioma cell sensitivity to TMZ by regulating the UTX/MGMT axis.

BAG1, MGMT, FOXO1, and DNAJA1 as potential drug targets for radiosensitizing cancer cell lines.

BACKGROUND AND PURPOSE: Activation of some signaling pathways can promote cell survival and have a negative impact on tumor response to radiotherapy. Here, the role of differences in expression levels of genes related to the poly(ADP-ribose) polymerase-1 (PARP-1), heat shock protein 90 (Hsp90), B-cell lymphoma 2 (Bcl-2), and phosphoinositide 3-kinase (PI3K) pathways in the survival or death of cells following X-ray exposure was investigated. METHODS: Eight human cell cultures (MCF-7 and MDA-MB-231: breast cancers; MCF-12A: apparently normal breast; A549: lung cancer; L132: normal lung; G28, G44 and G112: glial cancers) were irradiated with X-rays. The colony-forming and real-time PCR based on a custom human pathway RT2 Profiler PCR Array assays were used to evaluate cell survival and gene expression, respectively. RESULTS: The surviving fractions at 2 Gy for the cell lines, in order of increasing radioresistance, were found to be as follows: MCF-7 (0.200 ± 0.011), G44 (0.277 ± 0.065), L132 (0.367 ± 0.023), MDA-MB-231 (0.391 ± 0.057), G112 (0.397 ± 0.113), A549 (0.490 ± 0.048), MCF-12A (0.526 ± 0.004), and G28 (0.633 ± 0.094). The rank order of radioresistance at 6 Gy was: MCF-7 < L132 < G44 < MDA-MB-231 < A549 < G28 < G112 < MCF-12A. PCR array data analysis revealed that several genes were differentially expressed between irradiated and unirradiated cell cultures. The following genes, with fold changes: BCL2A1 (21.91), TP53 (8743.75), RAD51 (11.66), FOX1 (65.86), TCP1 (141.32), DNAJB1 (3283.64), RAD51 (51.52), and HSPE1 (12887.29) were highly overexpressed, and BAX (-127.21), FOX1 (-81.79), PDPK1 (-1241.78), BRCA1 (-8.70), MLH1 (-12143.95), BCL2 (-18.69), CCND1 (-46475.98), and GJA1 (-2832.70) were highly underexpressed in the MDA-MB-231, MCF-7, MCF-12A, A549, L132, G28, G44, and G112 cell lines, respectively. The radioresistance in the malignant A549 and G28 cells was linked to upregulation in the apoptotic, DNA repair, PI3K, and Hsp90 pathway genes BAG1, MGMT, FOXO1, and DNAJA1, respectively, and inhibition of these genes resulted in significant radiosensitization. CONCLUSIONS: Targeting BAG1, MGMT, FOXO1, and DNAJA1 with specific inhibitors might effectively sensitize radioresistant tumors to radiotherapy.

Epigenetic agents in combined anticancer therapy.

In the last decade, epigenetic drugs (such as inhibitors of DNA methyltransferases and histone deacetylases) have been intensively used for cancer treatment. Their applications have shown high anticancer effectivity and tolerable side effects. However, they are unfortunately not effective in the treatment of some types and phenotypes of cancers. Nevertheless, several studies have demonstrated that problems of drug efficacy can be overcome through the combined application of therapeutic modulates. Therefore, combined applications of epigenetic agents with chemotherapy, radiation therapy, immunotherapy, oncolytic virotherapy and hyperthermia have been presented. This review summarizes and discusses the general principles of this approach, as introduced and supported by numerous examples. In addition, predictions of the future potential applications of this methodology are included.

Epi-drugs in combination with immunotherapy: a new avenue to improve anticancer efficacy.

Immune checkpoint factors, such as programmed cell death protein-1/2 (PD-1, PD-2) or cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) receptors, are targets for monoclonal antibodies (MAbs) developed for cancer immunotherapy. Indeed, modulating immune inhibitory pathways has been considered an important breakthrough in cancer treatment. Although immune checkpoint blockade therapy used to treat malignant diseases has provided promising results, both solid and haematological malignancies develop mechanisms that enable themselves to evade the host immune system. To overcome some major limitations and ensure safety in patients, recent strategies have shown that combining epigenetic modulators, such as inhibitors of histone deacetylases (HDACi) or DNA methyltransferases (DNMTi), with immunotherapeutics can be useful. Preclinical data generated using mouse models strongly support the feasibility and effectiveness of the proposed approaches. Indeed, co-treatment with pan- or class I-selective HDACi or DNMTi improved beneficial outcomes in both in vitro and in vivo studies. Based on the evidence of a pivotal role for HDACi and DNMTi in modulating various components belonging to the immune system, recent clinical trials have shown that both HDACi and DNMTi strongly augmented response to anti-PD-1 immunotherapy in different tumour types. This review describes the current strategies to increase immunotherapy responses, the effects of HDACi and DNMTi on immune modulation, and the advantages of combinatorial therapy over single-drug treatment.

[Inactivation of PMS2 gene by promoter methylation in nasopharyngeal carcinoma].

Objective: To investigate the inactivation of PMS2 gene mediated by promoter methylation and its regulatory mechanism in nasopharyngeal carcinoma (NPC). Methods: Fifty-four NPC tissues, 16 normal nasopharyngeal epithelia (NNE), 5 NPC cell lines (CNE1, CNE2, TWO3, HNE1 and HONE1) and 1 normal nasopharyngeal epithelial cell line (NP69) were collected.Methylation-specific PCR (MSP) was used to detect the PMS2 promoter methylation, semi-quantitative reverse transcription PCR (qRT-PCR) was applied to determine its mRNA expression, and immunohistochemistry (IHC) was used to detect the protein expression of PMS2. The expressions of PMS2 mRNA in CNE1 and CNE2 cells before and after treated with methyltransferase inhibitor 5-aza-2-deoxycytidine were analyzed by qRT-PCR. The impact of methylation and demethylation on the mRNA expression of PMS2, and the association of mRNA and protein expression of PMS2 with clinicopathological features of nasopharyngeal cancer were analyzed. Results: Methylation of PMS2 gene was detected in all of the five NPC cell lines, but not in normal nasopharyngeal epithelial NP69 cells. The methylation rate of PMS2 gene in NPC tissues was 63% (34/54), significantly higher than that of the normal nasopharyngeal epithelia (0/16, P<0.001). The expression levels of PMS2 mRNA and protein were significantly down-regulated in the 54 NPC tissues when compared with those in the 16 NNE tissues (P<0.001), and were also significantly lower in the 34 methylated NPC tissues than those in the 20 unmethylated NPC tissues (P<0.001). After treatment with 5-aza-2-deoxycytidine, the expression of PMS2 mRNA was restored in the CNE1 and CNE2 cells.However, the expressions of PMS2 mRNA and protein were not significantly correlated with patients' age, gender, TNM stage, histopathologic type or lymph node metastasis (P>0.05 for all). Conclusions: Promoter methylation-mediated inactivation of PMS2 gene participates in carcinogenesis and development of NPC. PMS2 may be a candidate tumor suppressor in the treatment for patients with inactivation of PMS2 promoter methylation.

Targeting the Sonic Hedgehog-Gli1 Pathway as a Potential New Therapeutic Strategy for Myelodysplastic Syndromes.

The complex mechanistic array underlying the pathogenesis of myelodysplastic syndrome (MDS) is still unclear. Although dysregulations of different signaling pathways involved in MDS have been described, the identification of specific biomarkers and therapy targets remains an important task in order to establish novel therapeutic approaches. Here, we demonstrated that the Shh signaling pathway is active in MDS and correlated it with disease progression. Additionally, the knockdown of Gli1 significantly inhibited cell proliferation in vitro and in vivo. Gli1 silencing also induced apoptosis and G0/G1 phase arrest. Furthermore, Gli1 silencing enhanced the demethylating effect of 5-aza-2'-deoxycytidine on the p15 gene promoter and subsequently promoted its expression by inhibiting DNA methyltransferase 1(DNMT1). Our findings show that the Shh signaling pathway plays a role in the pathogenesis and disease progression of MDS, and proceeds by modulating DNA methylation. This pathway may prove to be a potential therapeutic target for enhancing the therapeutic effects of 5-azacytidine on malignant transformation of MDS.

DNA demethylating agent decitabine increases AQP5 expression and restores salivary function.

Xerostomia is the symptom of oral dryness resulting most frequently, but not exclusively, from salivary gland hypofunction. Because the prevalence of xerostomia may increase with age, it has multiple oral health consequences in aging populations. In the present study, we demonstrate that the in vivo administration of 5-aza-2'-deoxycytidine (5-Aza-CdR; decitabine), a DNA demethylating agent, to the murine aging model C57BL/6CrSlc mice (24 wks old) increased the volumes of salivary flow compared with those of control mice. Western blot analysis and immunohistochemical staining demonstrated the augmented expression of AQP5 protein in the salivary glands of 5-Aza-CdR-treated mice compared with those of control mice. In addition, AQP5 protein expression levels in 5-Aza-CdR-treated old mice (27 wks old) were much higher than those in untreated and young mice (6 wks old). Global methylation levels in the salivary glands were significantly lower in the 5-Aza-CdR-treated mice than in the untreated mice. Moreover, the induction of demethylation in the AQP5 promoter of 5-Aza-CdR-treated mice was stronger than in the control mice. Analysis of our data therefore suggests that a DNA demethylating agent may be a useful drug for restoring hyposalivation in elderly individuals, thereby leading to the resolution of xerostomia.

Methods in DNA methylation profiling.

Metastable and somatically heritable patterns of DNA methylation provide an important level of genomic regulation. In this article, we review methods for analyzing these genome-wide epigenetic patterns and offer a perspective on the ever-expanding literature, which we hope will be useful for investigators who are new to this area. The historical aspects that we cover will be helpful in interpreting this literature and we hope that our discussion of the newest analytical methods will stimulate future progress. We emphasize that no single approach can provide a complete view of the overall methylome, and that combinations of several modalities applied to the same sample set will give the clearest picture. Given the unexpected epigenomic patterns and new biological principles, as well as new disease markers, that have been uncovered in recent studies, it is likely that important discoveries will continue to be made using genome-wide DNA methylation profiling.

Temozolomide preferentially depletes cancer stem cells in glioblastoma.

The prognosis of patients suffering from glioblastoma (GBM) is dismal despite multimodal therapy. Although chemotherapy with temozolomide may contain tumor growth for some months, invariable tumor recurrence suggests that cancer stem cells (CSC) maintaining these tumors persist. We have therefore investigated the effect of temozolomide on CD133(+) and CD133(-) GBM CSC lines. Although differentiated tumor cells constituting the bulk of all tumor cells were resistant to the cytotoxic effects of the substance, temozolomide induced a dose- and time-dependent decline of the stem cell subpopulation. Incubation with sublethal concentrations of temozolomide for 2 days completely depleted clonogenic tumor cells in vitro and substantially reduced tumorigenicity in vivo. In O(6)-methylguanine-DNA-methyltransferase (MGMT)-expressing CSC lines, this effect occurred at 10-fold higher doses compared with MGMT-negative CSC lines. Thus, temozolomide concentrations that are reached in patients were only sufficient to completely eliminate CSC in vitro from MGMT-negative but not from MGMT-positive tumors. Accordingly, our data strongly suggest that optimized temozolomide-based chemotherapeutic protocols might substantially improve the elimination of GBM stem cells and consequently prolong the survival of patients.

Epigenetic combination therapy as a tumor-selective treatment approach for hepatocellular carcinoma.

BACKGROUND: Innovative epigenetic therapeutics comprise histone deacetylase inhibitors (HDAC-I) and demethylating agents (DA). It was recently found that HDAC-I compounds exhibit profound therapeutic activities against hepatocellular carcinoma (HCC). A comprehensive preclinical investigation was performed on the potential of a combined HDAC-I/DA epigenetic regimen for the highly chemotherapy-resistant HCC entity. METHODS: Human HCC-derived cell lines or primary human hepatocytes (PHH) were treated with HDAC-I compound suberoylanilide hydroxamic acid (SAHA) or DA compound 5-aza-2'-deoxycytidine (5-aza-dC) or both and examined for cellular damage, proliferation, histone acetylation pattern, and DNA methylation. In vivo activities were investigated in a xenograft hepatoma model. RESULTS: Monotherapeutic application of SAHA or 5-aza-dC was found to induce substantial antiproliferative effects in HCC-derived cells, strongly enhanced by combined SAHA and 5-aza-dC treatment. PHH from different human donors did not exhibit any relevant cellular damage even when applying high doses of the combination regimen, whereas HCC-derived cell lines showed a dose-dependent damage. In vivo testing demonstrated a statistical significant inhibition of hepatoma cell growth for the combined treatment regime. CONCLUSIONS: Because the combined HDAC-I/DA epigenetic approach was found to produce significant antitumor effects in HCC model systems and did not impair cellular integrity of untransformed hepatocytes, this combination therapy is now considered for further investigation in clinical trials.

Lytic induction and apoptosis of Epstein-Barr virus-associated gastric cancer cell line with epigenetic modifiers and ganciclovir.

Epstein-Barr virus (EBV) is an oncogenic herpes virus. EBV gene transcription is regulated by an epigenetic mechanism to establish a persistent infection and to evade the host immune system. We found that low concentrations of epigenetic modifying agents, 5-aza-2'-deoxycytidine (5-aza-CdR) or trichostatin A (TSA), induced the expression of BMRF1, BZLF1, and BRLF1 genes, which are found in the lytic form of the virus, in an EBV-positive gastric cancer cell line. This effect did not involve PI3 kinase, MAP/ERK kinase, protein kinase C delta, or p38 MAPK signaling pathway. The cytotoxic effect of ganciclovir (GCV) was enhanced after the lytic induction by epigenetic modifiers, and the combination of GCV and epigenetic modifiers induced apoptosis, which is dependent on caspases. In conclusion, the combination of GCV with 5-aza-CdR or TSA might be a useful therapeutic strategy for EBV-induced human gastric cancer.

Overcoming resistance to interferon-induced apoptosis of renal carcinoma and melanoma cells by DNA demethylation.

Epigenetic editing of gene expression by aberrant methylation of DNA may help tumor cells escape attack from the innate and acquired immune systems. Resistance to antiproliferative effects and apoptosis induction by interferons (IFNs) was postulated to result from silencing of IFN response genes by promoter hypermethylation. Treatment of human ACHN renal cell carcinoma (RCC) and A375 melanoma cells with the DNA demethylating nucleoside analog 5-AZA-2'-deoxycytidine (5-AZA-dC) synergistically augmented antiproliferative effects of IFN- alpha (alpha) 2 and IFN-beta (beta). Either 5-AZA-dC or an antisense to DNA methyltransferase 1 (DNMT1) overcame resistance to apoptosis induction by IFNs with up to 85% apoptotic cells resulting from the combinations. No similar potentiation occurred in normal kidney epithelial cells. IFN response genes were augmented more than 10 times in expression by 5-AZA-dC. Demethylation by 5-AZA-dC of the promoter of the prototypic, apoptosis-associated IFN response gene XAF1 was confirmed by methylation-specific polymerase chain reaction. siRNA to XAF1 inhibited IFN-induced apoptosis; conversely, overexpression of XAF1 overcame resistance to apoptosis induction by IFN-beta. As occurred with apoptosis-resistant melanoma cells in vitro, tumor growth inhibition in the nude mouse of human A375 melanoma xenografts resulted from treatment with 5-AZA-dC in combination with IFN-beta, an effect not resulting from either single agent. The importance of epigenetic remodeling of expression of immune-modifying genes in tumor cells was further suggested by identifying reactivation of the cancer-testis antigens MAGE and RAGE in ACHN cells after DNMT1 depletion. Thus, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumor-associated antigens.

The prospective role of abnormal methyl metabolism in cadmium toxicity.

Several lines of evidence point to the probable role of abnormal methylation processes in the toxicology of metals and other xenobiotics. The spectrum of toxic effects exhibited by such metals as Ni, As, and Cd, as well as by Zn deficiency, often resemble those seen in animals chronically fed methyl-deficient diets. These metal-associated pathologies include cancer, atherosclerosis, birth defects, neurological disturbances, and pancreatic lesions. In addition, each of the above agents has been shown to alter normal methyl group metabolism in vivo or in vitro. In the present studies, we compared the effects on the enzyme DNA methyltransferase (MTase) of two metal ions: the essential metal Zn and the carcinogen Cd. MTase extracts were obtained from the hepatic nuclei of rats fed a methyl-deficient diet (lacking choline and folate) for 7 and 24 weeks. Control animals were fed the same diet supplemented with each of these vitamins. Zn and Cd both inhibited MTase in the nuclear extracts from both the control and the methyl-deficient rats. The inhibitory activity of Cd was greater than that of Zn regardless of whether the nuclear extracts were from the control or the deficient animals. In addition, the kinetics of Cd inhibition of MTase activity were different in the nuclear extracts from the control and methyl-deficient rats. The results provide evidence that the carcinogenic effects of Cd may be mediated in part through abnormal DNA methylation.

Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer.

Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.