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

Zambrano P, Segura-Pacheco B, Perez-Cardenas E, Cetina L, Revilla-Vazquez A, Taja-Chayeb L, Chavez-Blanco A, Angeles E, Cabrera G, Sandoval K, Trejo-Becerril C, Chanona-Vilchis J, Duenas-González A - BMC Cancer (2005)

Bottom Line: Hydralazine was well tolerated.There was neither change in the methylation status of H19 and clone 1.2 nor changes in global DNA methylation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología/Instituto de Investigaciones Biomédicas, UNAM, Mexico. pila_55@yahoo.com

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.

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Related in: MedlinePlus

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
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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

Mentions: Methylation analysis of the biopsies taken before and after 10 days of hydralazine administration was performed in all 16 patients. Methylation results involving the genes analyzed were variable. Overall, 70% (89 out of 128) of the pretreatment samples analyzed (8 genes for each of the 16 pre-treatment biopsies) had at least one methylated gene, and all 16 patients had at least one methylated gene. Analysis by individual genes showed the following rates of methylated genes: APC (94%), ER (25%) FHIT (88%), GSTp1 (88%), MGMT(81%), p16(19%), RARβ (62%), and DAPK (100%), irrespective of the dose of hydralazine used, the post-treatment biopsies showed a variable demethylation rate according to the gene, varying from 15% (2/13 samples) for the MGMT gene, to 67% of demethylation for the p16 gene (2 out of 3 samples), (Figure 1A). Representative cases of the results are shown in Figure 1B. Correlation between demethylation analysis and dose level revealed the following results: at 50 mg a day, 40% of methylated genes suffered demethylation; at 75 mg it was 52%, at 100 mg dose the rate was 43%, and 32% for the 150 mg dose (Figure 1C).


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

Zambrano P, Segura-Pacheco B, Perez-Cardenas E, Cetina L, Revilla-Vazquez A, Taja-Chayeb L, Chavez-Blanco A, Angeles E, Cabrera G, Sandoval K, Trejo-Becerril C, Chanona-Vilchis J, Duenas-González A - BMC Cancer (2005)

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
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1131894&req=5

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
Mentions: Methylation analysis of the biopsies taken before and after 10 days of hydralazine administration was performed in all 16 patients. Methylation results involving the genes analyzed were variable. Overall, 70% (89 out of 128) of the pretreatment samples analyzed (8 genes for each of the 16 pre-treatment biopsies) had at least one methylated gene, and all 16 patients had at least one methylated gene. Analysis by individual genes showed the following rates of methylated genes: APC (94%), ER (25%) FHIT (88%), GSTp1 (88%), MGMT(81%), p16(19%), RARβ (62%), and DAPK (100%), irrespective of the dose of hydralazine used, the post-treatment biopsies showed a variable demethylation rate according to the gene, varying from 15% (2/13 samples) for the MGMT gene, to 67% of demethylation for the p16 gene (2 out of 3 samples), (Figure 1A). Representative cases of the results are shown in Figure 1B. Correlation between demethylation analysis and dose level revealed the following results: at 50 mg a day, 40% of methylated genes suffered demethylation; at 75 mg it was 52%, at 100 mg dose the rate was 43%, and 32% for the 150 mg dose (Figure 1C).

Bottom Line: Hydralazine was well tolerated.There was neither change in the methylation status of H19 and clone 1.2 nor changes in global DNA methylation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología/Instituto de Investigaciones Biomédicas, UNAM, Mexico. pila_55@yahoo.com

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.

Show MeSH
Related in: MedlinePlus