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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

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.
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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.

Mentions: All cases showed expression of β actin. Gene expression analysis showed that 90% (116 out of 128) of the tumor samples expressed the messenger in the pre-treatment and post-treatment biopsies regardless of methylation status, hence were not informative. On the other hand, there were only 12 informative cases. Of these 12 cases, three (25%) (having only the methylated band pretreatment) showed no re-expression after treatment (2/ DAPK, 1/GSTp1 cases). These three cases received the 50 mg dose. In the remaining 9 cases (75%) expression of the gene was re-induced after treatment. These nine cases behaved as follows: Five cases were RT-PCR negative pretreatment (only methylated band) and converted positive post-treatment, displaying methylated and unmethylated bands (FHIT two cases -100 and 150 mg doses- MGMT two cases -75 and 150 mg doses, GSTp1 one case -50 mg dose). Three cases RT-PCR negative pre-treatment with methylated band which converted to unmethylated and expression positive in the post-therapy biopsy (DAPK and ER genes at 75 mg, GSTp1 at 150 mg dose), and finally, a RT-PCR negative in the pre-treatment biopsy despite having methylated and unmethylated bands which converted to RT-PCR positive accompanied by only unmethylated band (GSTp1 case at 75 mg). Table 5, Figure 2D. Three representative cases are shown in Figure 2A, B, C.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: All cases showed expression of β actin. Gene expression analysis showed that 90% (116 out of 128) of the tumor samples expressed the messenger in the pre-treatment and post-treatment biopsies regardless of methylation status, hence were not informative. On the other hand, there were only 12 informative cases. Of these 12 cases, three (25%) (having only the methylated band pretreatment) showed no re-expression after treatment (2/ DAPK, 1/GSTp1 cases). These three cases received the 50 mg dose. In the remaining 9 cases (75%) expression of the gene was re-induced after treatment. These nine cases behaved as follows: Five cases were RT-PCR negative pretreatment (only methylated band) and converted positive post-treatment, displaying methylated and unmethylated bands (FHIT two cases -100 and 150 mg doses- MGMT two cases -75 and 150 mg doses, GSTp1 one case -50 mg dose). Three cases RT-PCR negative pre-treatment with methylated band which converted to unmethylated and expression positive in the post-therapy biopsy (DAPK and ER genes at 75 mg, GSTp1 at 150 mg dose), and finally, a RT-PCR negative in the pre-treatment biopsy despite having methylated and unmethylated bands which converted to RT-PCR positive accompanied by only unmethylated band (GSTp1 case at 75 mg). Table 5, Figure 2D. Three representative cases are shown in Figure 2A, B, C.

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