A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes

Abstract

Background: The antihypertensive compound hydralazine is a known demethylating agent. This phase I study evaluated the tolerability and its effects upon DNA methylation and gene reactivation in patients with untreated cervical cancer.

Methods: Hydralazine was administered to cohorts of 4 patients at the following dose levels: I) 50 mg/day, II) 75 mg/day, III) 100 mg/day and IV) 150 mg/day. Tumor biopsies and peripheral blood samples were taken the day before and after treatment. The genes APC, MGMT; ER, GSTP1, DAPK, RARbeta, FHIT and p16 were evaluated pre and post-treatment for DNA promoter methylation and gene expression by MSP (Methylation-Specific PCR) and RT-PCR respectively in each of the tumor samples. Methylation of the imprinted H19 gene and the "normally methylated" sequence clone 1.2 was also analyzed. Global DNA methylation was analyzed by capillary electrophoresis and cytosine extension assay. Toxicity was evaluated using the NCI Common Toxicity Criteria.

Results: Hydralazine was well tolerated. Toxicities were mild being the most common nausea, dizziness, fatigue, headache and palpitations. Overall, 70% of the pretreatment samples and all the patients had at least one methylated gene. Rates of demethylation at the different dose levels were as follows: 50 mg/day, 40%; 75 mg/day, 52%, 100 mg/day, 43%, and 150 mg/day, 32%. Gene expression analysis showed only 12 informative cases, of these 9 (75%) re-expressed the gene. There was neither change in the methylation status of H19 and clone 1.2 nor changes in global DNA methylation.

Conclusion: Hydralazine at doses between 50 and 150 mg/day is well tolerated and effective to demethylate and reactivate the expression of tumor suppressor genes without affecting global DNA methylation.

Figure 1

1A. 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. 1B. Representative cases of genes (M methylated, U unmethylated; pre/post): M/M; M/U; U/U, M/U; MU/U; M/ U-M. 1C. 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

Figure 2

Representative cases correlating methylation and re-expression before and after hydralazine treatment. 2A is a patient treated with 75 mg/day that demethylated and re-expressed the DAPK gene. 2B 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. 2C is a 50 mg/day patient which failed to demethylate the DAPK gene and therefore lacked expression. 2D 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.

Figure 3

3A. Photomicrography of a representative set of pre and post-treatment tumor biopsies showing that the malignant component represents almost the half of the tumor. 3B. Methylation analysis of the DAPK gene in the four fragments of the tumor biopsy of an untreated cervical cancer patient. Despite all fragments contained different proportions of malignant cells and stroma the four samples show methylated and unmethylated bands.

Figure 4

DNA methylation analysis obtained from peripheral mononuclear cells of genes "imprinted or normally methylated". Clone 1.2 remained methylated in all cases whereas for the imprinted H19 gene, the pattern of U and M alleles did not change.

Figure 5

5A. Capillary electrophoretic analysis of global methylation. Relative methylation showed no variation in percentage of ^mC after treatment (37.3% versus 36.3%). 5B is a electropherogram showing the separation of C and ^mC. 5C is the percent increase in radiolabeled incorporation pre and post-treatment as compared to the control of undigested DNA.