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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

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.
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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.

Mentions: Global tumor DNA methylation was evaluated by two methods, capillary electrophoresis and cytosine extension. Enough DNA to perform capillary electrophoresis was only available in five cases (four patients taking 100 mg, and one taking 150 mg/day respectively). The relative methylation of each DNA sample was taken as the percentage of dmC in total cytosine. Figure 5A shows the relative methylation in theses cases. Remarkably, relative methylation in all cases was between 34.2 and 38.4% and there was no change in methylation levels after hydralazine treatment (37.3% ± 0.81 and 36.3% ± 1.1 pre and post-treatment respectively). An electropherogram showing the separation of the analytes of interest, C and mC peak are shown in Figure 5B. In the cytosine extension assay, the extent of [(3)H]dCTP incorporation after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG sites. The results of this assay showed no statistically significant changes in the tumor samples pre and post-treatment as the percentage increase in radiolabel incorporation were 15% ± 31.3% and 7% ±16.7% respectively, Figure 5C.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Global tumor DNA methylation was evaluated by two methods, capillary electrophoresis and cytosine extension. Enough DNA to perform capillary electrophoresis was only available in five cases (four patients taking 100 mg, and one taking 150 mg/day respectively). The relative methylation of each DNA sample was taken as the percentage of dmC in total cytosine. Figure 5A shows the relative methylation in theses cases. Remarkably, relative methylation in all cases was between 34.2 and 38.4% and there was no change in methylation levels after hydralazine treatment (37.3% ± 0.81 and 36.3% ± 1.1 pre and post-treatment respectively). An electropherogram showing the separation of the analytes of interest, C and mC peak are shown in Figure 5B. In the cytosine extension assay, the extent of [(3)H]dCTP incorporation after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG sites. The results of this assay showed no statistically significant changes in the tumor samples pre and post-treatment as the percentage increase in radiolabel incorporation were 15% ± 31.3% and 7% ±16.7% respectively, Figure 5C.

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