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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

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.
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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.

Mentions: Because the sampling of tumor may lead to tissue specimens with different degree of "contamination" with non-malignant that may affect the result of methylation in the post-treatment, biopsies, all samples were analyzed by a pathologist. The results showed that in all biopsy samples the content of malignant cells varied from 30 to 70%. A representative set is shown in Figure 3A. In addition, we reasoned that if tumor heterogeneity were of a magnitude to yield different methylation patterns in the tumors regardless of the treatment, then multisampling a tumor with subsequent methylation analysis of each of the tumor fragments will render zones with different methylation pattern. The analysis of the surgical specimen of an untreated patient showed that the methylation of the DAPK gene promoter was the same in the four fragments of the tumor. Figure 3B.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Because the sampling of tumor may lead to tissue specimens with different degree of "contamination" with non-malignant that may affect the result of methylation in the post-treatment, biopsies, all samples were analyzed by a pathologist. The results showed that in all biopsy samples the content of malignant cells varied from 30 to 70%. A representative set is shown in Figure 3A. In addition, we reasoned that if tumor heterogeneity were of a magnitude to yield different methylation patterns in the tumors regardless of the treatment, then multisampling a tumor with subsequent methylation analysis of each of the tumor fragments will render zones with different methylation pattern. The analysis of the surgical specimen of an untreated patient showed that the methylation of the DAPK gene promoter was the same in the four fragments of the tumor. Figure 3B.

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