Hydralazine target: from blood vessels to the epigenome - PubMed (original) (raw)

Hydralazine target: from blood vessels to the epigenome

Claudia Arce et al. J Transl Med. 2006.

Abstract

Hydralazine was one of the first orally active antihypertensive drugs developed. Currently, it is used principally to treat pregnancy-associated hypertension. Hydralazine causes two types of side effects. The first type is an extension of the pharmacologic effect of the drug and includes headache, nausea, flushing, hypotension, palpitation, tachycardia, dizziness, and salt retention. The second type of side effects is caused by immunologic reactions, of which the drug-induced lupus-like syndrome is the most common, and provides clues to underscoring hydralazine's DNA demethylating property in connection with studies demonstrating the participation of DNA methylation disorders in immune diseases. Abnormalities in DNA methylation have long been associated with cancer. Despite the fact that malignant tumors show global DNA hypomethylation, regional hypermethylation as a means to silence tumor suppressor gene expression has attracted the greatest attention. Reversibility of methylation-induced gene silencing by pharmacologic means, which in turns leads to antitumor effects in experimental and clinical scenarios, has directed efforts toward developing clinically useful demethylating agents. Among these, the most widely used comprise the nucleosides 5-azacytidine and 2'deoxy-5-azacytidine; however, these agents, like current cytotoxic chemotherapy, causes myelosuppression among other side effects that could limit exploitation of their demethylating properties. Among non-nucleoside DNA demethylating drugs currently under development, the oral drug hydralazine possess the ability to reactivate tumor suppressor gene expression, which is silenced by promoter hypermethylation in vitro and in vivo. Decades of extensive hydralazine use for hypertensive disorders that demonstrated hydralazine's clinical safety and tolerability supported its testing in a phase I trial in patients with cancer, confirming its DNA demethylating activity. Hydralazine is currently being evaluated, along with histone deacetylase inhibitors either alone or as adjuncts to chemotherapy and radiation, for hematologic and solid tumors in phase II studies.

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Figures

Figure 1

Figure 1

Methylation analysis of the ER, RARβ, and p16 genes. A, G is undigested genomic DNA as positive control, H is HpaII, and M is MspI. The band in H of control lanes indicates lack of digestion with HpaII and, therefore, methylation of untreated MDA-231 cells. B and C show the analysis by methylation-specific PCR of RARβ and p16 genes in MCF-7 and T24 cells. W is wild type, M is methylated primers, and U is unmethylated primers. D and E show a time course experiment of RARβ and p16 gene demethylation over 5 days on the MCF-7 and T24 cells, respectively. (Reproduced with permission from the AACR. Ref. 83. Clin Cancer Res 2003, 9:1596–1603).

Figure 2

Figure 2

Expression of the mRNA and proteins of A, ER (MDA-231); B, RARβ (MCF-7); and C, p16 (T24) genes by RT-PCR and Western blot. The positive control (+ctr) for A is MCF-7, for B is MDA-231 exposed to 5-aza-CdR/atRA, and for C is HeLa cells. (Reproduced with permission from the AACR. Ref. 83. Clin Cancer Res 2003, 9:1596–1603).

Figure 3

Figure 3

Analysis of methylation and product expression of the ER gene in nude mice. A shows that the tumor of control mice (CTR lanes) was methylated as shown by the band in the Lane H indicating no digestion with HpaII. The band disappeared with the treatment with 5-aza-CdR and hydralazine, which indicates gene demethylation. In PROC (procainamide) lanes, the band is absent in MspI but is very weak in HpaII, indicating that was only partially demethylated; B and D are the product expression by RT-PCR and Western blot. In both cases, the intensity of bands in procainamide is weaker, correlating with a partial demethylation, (C and E are the loading controls, GAPDH and actine respectively). G is undigested genomic DNA. (Reproduced with permission from the AACR. Ref. 83. Clin Cancer Res 2003, 9:1596–1603).

Figure 4

Figure 4

Docking between the enzyme DNA methyltransferase and hydralazine showing the residues at the pocket (Lys 162 and Arg 165) that interact with the hydralazine molecule (Modified from ref. 84. Lett Drug Design Discov 2005, 2:282–286).

Figure 5

Figure 5

In in vitro methyltransferase assay of hydralazine. There was no digestion with HpaII (lanes H, with 0 and 5 μM of hydralazine) indicating full methylation. Starting at 10 μM of hydralazine, bands in the lanes digested with HpaII (H) are visible, and at 20 μM the pattern of bands in HpaII (H) and MspI (M) digestions is similar indicating full demethylation. (MW: molecular weight). The substrate DNA for the in vitro methylation assay was a 1112 bp fragment of the type I Human Herpes Simplex virus tymidine kinase gene which has a high GC content. The methylation reaction contained 1 μg of substrate DNA and 10 units of M.SssI methylase (0.5 μmol/L, New England Biolabs, Beverly, MA) in a final volume of 30 μL. Hydralazine was added to final concentrations of 0, 5, 10, 15, and 20 μmol/L starting two hours before adding the M.SssI enzyme for the methylation reaction. Reactions were done at 37°C for 2 hours. After completion, the reaction the DNA was purified using the rapid PCR Purification system (Ijamsville, MD). Then, equal volumes of extracted DNA for each sample were placed in two separated eppendorf tubes for restriction enzyme digestion (one tube with HpaII and the other with MspI) both from New England Biolabs. Each reaction contained 10 U of the enzyme, in a final reaction volume of 50 μL at 37° for 3 hours. After digestion, samples were reduced to dryness by speedvac concentration and redissolved in 10 μL of ddH20 and then loaded into a 2% Tris-borate EDTA agarose gels for electrophoresis.

Figure 6

Figure 6

Expression of DNMT1 and DNMT3a in MCF-7 cells. The expression of DNMT1 and DNMT3a mRNA is decreased in MCF-7 cells treated with hydralazine. The lower band intensity was confirmed by densitometric (right) analysis, (control is untreated cells). MCF-7 cells were growth arrested for 48 hours by serum deprivation, then treated for 24 hours with hydralazine at 10 μM and then RNA extracted for analysis. Reverse transcription was carried out as previously described [110] using random hexamers, superscript II reverse transcriptase (Life Technologies) and 2.5 μg of total RNA as recommended by the manufacturer in a total volume of 50 μL. One microliter of RT reaction was used for subsequent PCR amplification for each of the desired transcripts with dNTPs and Amplitaq (Applied Biosystems). Primer sequences used were as follows: DNMT1 sense 5-gat cga att cat gcc ggc gcg tac cgc ccc ag-3 and antisense 5-atg gtg gtt tgc ctg gtg c-3. DNMT3a sense 5-ggg gac gtc cgc agc gta cac-3 and antisense 5-cag ggt tgg act cga gaa atc g-3. Amplification conditions were: 94 C° for 5 min 1 cycle; 94 C° 30 sec, (58°C for DNMT1 and 65°C for DNMT3a) annealing temperature for 1 min, and 72 C° for 30 sec, 35 cycles.

Figure 7

Figure 7

HeLa cells were treated for 24 hours with panel a: cisplatin (cddp), panel b: adriamycin (adr) or panel c: gemcitabine (gem) at or close to IC50 plus hydralazine (h) and valproic acid (v). Afterwards medium was removed and fresh medium containing only hydralazine and valproic acid was added for additional 48 hours after which viability was measured. There was a significant higher cytotoxicity of the chemotherapeutic agent when used in combination with hydralazine plus valproic acid. Cisplatin alone at 12 μM caused a reduction in viability to 37% in HeLa cells which was essentially zero viability when cells were co-treated with hydralazine plus valproic acid (h-v-c). The increasing toxicity of the combination was also seen with adriamycin and gemcitabine. Viability was 42% and 45% in the chemotherapy drugs alone versus 27% and 37% respectively when hydralazine plus valproic acid were added to chemotherapy drugs (h-v-c). (Reproduced from ref. 89. Cancer Cell Int 2006,6:2).

Figure 8

Figure 8

A. Pre- (dark bars) and post-hydralazine treatment (light bars). The bars represent the number of patients that showed methylation for each studied gene from each of the 16 patients. B. Representative cases of genes (M methylated, U unmethylated; pre/post): M/M; M/U; U/U, M/U; MU/U; M/ U-M. C. Percentage of demethylation after treatment according to the dose. Percentage was calculated considering 100% methylation the total number of pre-treatment methylated genes in each cohort of 4 patients (Reproduced from ref. 74. BMC Cancer 2005, 5:44).

Figure 9

Figure 9

Representative cases correlating methylation and re-expression before and after hydralazine treatment. A is a patient treated with 75 mg/day that demethylated and re-expressed the DAPK gene. B corresponds to a patient receiving 150 mg/day who showed only the methylated band pre-treatment, but both bands after treatment, which correlated with re-expression of MGMT. C is a 50 mg/day patient which failed to demethylate the DAPK gene and therefore lacked expression. D represents the distribution of informative cases. From the 128 genes/cases, 116 were RT-PCR positive regardless of the methylation status, hence were not informative. In the remaining 12 cases, nine demethylated and re-expressed the gene. (Reproduced from ref. 74. BMC Cancer 2005, 5:44).

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