PROCAINA 2Ana Villar-Garea, Mario F. Fraga, Jesus Espada, and Manel Esteller
Cancer Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Centre (CNIO), Madrid 28029, Spain

Published in: CANCER RESEARCH 63, 4984–4989, August 15, 2003

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Methylation-associated silencing of tumor suppressor genes is recognized as being a molecular hallmark of human cancer. Unlike genetic alterations, changes in DNA methylation are potentially reversible. This possibility has attracted considerable attention from a therapeutics standpoint.
Nucleoside-analogue inhibitors of DNA methyltransferases, such as 5-aza-2-deoxycytidine, are able to demethylate DNA and restore silenced gene expression. Unfortunately, the clinical utility of these compounds has not yet been fully realized, mainly because of their side effects. A few  non-nucleoside inhibitors of DNA methyltransferases have been reported, including the anti-arrhythmia drug procainamide. Following this need to find new demethylating agents, we have tested the potential use of procaine, an anesthetic drug related to procainamide. Using the MCF-7 breast cancer cell line, we have found that procaine is a DNA-demethylating agent that produces a 40% reduction in 5-methylcytosine DNA content as determined by high-performance capillary electrophoresis or total DNA enzyme digestion. Procaine can also demethylate densely hypermethylated CpG islands, such as those located in the promoter region of the RAR2 gene, restoring gene expression of epigenetically silenced genes. This property may be explained by our finding that procaine binds to CpG-enriched DNA. Finally, procaine also has growth-inhibitory effects in these cancer cells, causing mitotic arrest. Thus, procaine is a promising candidate agent for future cancer therapies based on epigenetics.

The inactivation of tumor suppressor genes is now recognized as being a major feature of all forms of human cancer. The re-expression in tumor cells of many of these genes can lead to suppression of cell growth (1, 2). Many of the demethylating agents are small versatile molecules that are in sharp contrast to the challenges of delivering gene therapy. As more methylation-mediated silenced genes are found in human neoplasms, there is increasing interest in the search for new demethylating agents of potential utility in cancer therapy. To this list, we may now add PCA, a drug that has been administered safely as a local anesthetic for many years.
We have found that PCA causes global DNA hypomethylation,demethylation and re-expression of a CpG-island-associated gene (RAR2), and growth inhibition in breast cancer cells. In this way, it behaves very similarly to procainamide (both molecules are 4-aminobenzoic acid derivates), which restores the expression of the hypermethylated GSTP1 gene in prostate cancer cells and diminishes xenograft tumor growth (11). Preclinical studies are now needed to ascertain whether PCA, in a similar manner to that of the classical demethylating agent DAC, synergizes with histone deacetylase inhibitors in the reactivation of dormant genes (29). One interesting aspect from a clinical standpoint is our observation that PCA stops the growth of cancer cells “in vitro.” This observation can explain why PCA increases the antitumoral activity of several conventional anticancer drugs, such as cisplatin, mitomycin C, peplomycin, and doxorubicin (30–33). Other conventional strategies for cancer treatment could also benefit from the newly identified hypomethylating and growth-inhibiting actions of PCA. This is the case in radiotherapy, in which PCA has been shown to radiosensitize hypoxic cells and to increase their hyperthermic killing (34, 35).
Until now, one of the limitations of DNA hypomethylating agents such as DAC in the clinical setting has been the side effects (mainly myelotoxicity) of the treatments and the concern that its incorporation into genomic DNA might lead to mutations (36). These setbacks are characteristic of all nucleoside analogues in general, not only DNAmethyltransferase inhibitors. PCA is not incorporated into the DNA but, instead, binds to DNA. Thus, PCA may be an example of an agent that demethylates DNA and reactivates methylated genes with less potential side effects. It is important to mention that the doses of PCA that achieve significant demethylation and growth-inhibitory effects in our study are of the same order as those administered in conjunction with antineoplastic drugs (30–33) or radiotherapy (34, 35). Most important, PCA has even proved to protect against chemotherapyrelated nephrotic and hepatic toxicities (31).
Our study supports a role for PCA as a promising DNA-hypomethylating drug with growth-inhibitory effects in cancer cells. Its long-established and safe use as a local anesthetic, with well-known pharmacological characteristics, may stimulate its prompt transition to preclinical and early clinical trials for epigenetics-based cancer treatments.