ZHI GAO (1,3), ZHIDONG XU (3), MING-SZU HUNG (3,4), YU-CHING LIN (4), TIANYOU WANG (1), MIN GONG (1), XIUYI ZHI (2), DAVID M. JABLONS (3) and LIANG YOU (3)
(1) Department of Cardiothoracic Surgery, Beijing Friendship Hospital
(2) Beijing Xuanwu Hospital, Capital Medical University, Beijing, P.R. China;
(3) Thoracic Oncology Lab., Dep. of Surgery, UCSF Comprehensive Cancer Center, Univ. of California, San Francisco, USA;
(4) Dep. of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Chiayi, Taiwan, R.O.C.
Published in: ONCOLOGY REPORTS 22: 1479-1484, 2009
ABSTRACT
Secreted Wingless type (Wnt) ligands have previously been shown to be involved in tumor developmental processes and oncogenesis. Aberrant promoter methylation of Wnt inhibitory factor-1 (WIF-1) is a fundamental mechanism of epigenetic silencing in human cancers. Procaine, a local anesthetic drug, and procainamide, a drug for the treatment of cardiac arrhythmias, have been reported as inhibitors of DNA methylation, causing demethylation and reactivation of methylation-silenced genes such as RAR-B and GSTP1. The promoter demethylation of WIF-1 has not previously been reported on. We demonstrated previously that WIF-1 is silenced due to promoter hypermethylation in lung cancer cell lines. In this study, we demonstrate promoter demethylation of WIF-1; restoration of WIF-1 expression, and underexpression of cytosolic B-catenin protein and TCF reporter activity, after procaine and procainamide treatment in H460 and A549 cell lines. Our results provide the first evidence that procaine and procainamide reactivate WIF-1 in these cancer cells and downregulate the Wnt canonical pathway. These results further suggest that procaine and procainamide may have a potential use for preventing the development of lung cancer.
DISCUSSION
Wnt signaling has emerged as a critical pathway in lung carcinogenesis. This has been amply demonstrated in numerous cancers (28,29). Several Wnt pathway proteins, including Wnt-1, Dvl-3 and B-catenin, have also been shown to be overexpressed in a number of cancers.
Wnt inhibitory factor-1 (WIF-1), a highly conserved gene that was first identified in the human retina, is a Wnt antagonist that inhibits Wnt signaling by direct binding to Wnt molecules. WIF-1 downregulates the Wnt pathway and inhibits NSCLC cell growth. Transfection with WIF-1 induces apoptosis in NSCLC cells and inhibits colony formation. Transfection by injecting WIF-1 plasmid also inhibits NSCLC tumor xenograft growth (16).
A growing list of genes have been identified that evidence abnormal CpG island promoter methylation (30). Methylation of the CpG island within the functional promoter region of WIF-1 is an important mechanism of aberrant Wnt signaling activation in cancer. Thus, by using methylation specific PCR and sequence analysis after bisulfite treatment, we demonstrated the frequent hypermethylation of the CpG islands in the functional WIF-1 promoter region, the hypermethylation correlated with their transcriptional silencing in human lung cancer cell lines (16). Conversely, promoter demethylation of WIF-1 restored the silenced WIF-1.
Procaine has been previously reported, in breast cancer cells, to cause global DNA hypomethylation, growth inhibition, and demethylation and re-expression of a methylation-silenced RAR-B2 (20). As such, procaine behaves very similarly to procainamide, the latter which specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1) (31), restores the expression of the hypermethylated GSTP1 gene in prostate cancer cells, and diminishes xenograft tumor growth (23). Therefore, we hypothesize that procaine and procainamide can also reactivate methylation-silenced WIF-1 gene.
In this study, we demonstrated that untreated H460 and A549 cells which were methylated silenced the expression of the WIF-1 gene. However, after exposure to procaine and procainamide, this epigenetic change was reversed. We also showed that H460 and A549 cells treated with procaine and procainamide decreased Tcf/Lef activity and expression of Bcatenin using a TOP/FOP Dual-Luciferase reporter assay and Western blot analysis, respectively.
These results are consistent with our hypothesis that procaine and procainamide may, in addition to restoring the methylated WIF-1 by demethylation, downregulate the Wnt canonical pathway in H460 and A549 cell lines.
The use of the nucleoside analogs such as DAC in clinical trials has been limited due to their side effects, including thrombocytopenia and neutropenia, probably caused by cytotoxic effects associated with the incorporation of the drugs into DNA, apart from their DNA-hypomethylation value.
There is thus an urgent need to discover less toxic demethylating agents that are not incorporated into DNA. Procaine and procainamide have been proposed as non-nucleosideinhibitors of DNA methylation. Their side effects are expected to be less as seen in nucleoside DNA methylation inhibitors because of their widespread clinical use (32).
We provided the first evidence that procaine and procainamide reactivate WIF-1 from a previously silenced methylation state and downregulate the canonical Wnt pathway. Procaine and procainmide may have a potential use for preventing the development of cancer.
Secreted Wingless type (Wnt) ligands have previously been shown to be involved in tumor developmental processes and oncogenesis. Aberrant promoter methylation of Wnt inhibitory factor-1 (WIF-1) is a fundamental mechanism of epigenetic silencing in human cancers. Procaine, a local anesthetic drug, and procainamide, a drug for the treatment of cardiac arrhythmias, have been reported as inhibitors of DNA methylation, causing demethylation and reactivation of methylation-silenced genes such as RAR-B and GSTP1. The promoter demethylation of WIF-1 has not previously been reported on. We demonstrated previously that WIF-1 is silenced due to promoter hypermethylation in lung cancer cell lines. In this study, we demonstrate promoter demethylation of WIF-1; restoration of WIF-1 expression, and underexpression of cytosolic B-catenin protein and TCF reporter activity, after procaine and procainamide treatment in H460 and A549 cell lines. Our results provide the first evidence that procaine and procainamide reactivate WIF-1 in these cancer cells and downregulate the Wnt canonical pathway. These results further suggest that procaine and procainamide may have a potential use for preventing the development of lung cancer.
DISCUSSION
Wnt signaling has emerged as a critical pathway in lung carcinogenesis. This has been amply demonstrated in numerous cancers (28,29). Several Wnt pathway proteins, including Wnt-1, Dvl-3 and B-catenin, have also been shown to be overexpressed in a number of cancers.
Wnt inhibitory factor-1 (WIF-1), a highly conserved gene that was first identified in the human retina, is a Wnt antagonist that inhibits Wnt signaling by direct binding to Wnt molecules. WIF-1 downregulates the Wnt pathway and inhibits NSCLC cell growth. Transfection with WIF-1 induces apoptosis in NSCLC cells and inhibits colony formation. Transfection by injecting WIF-1 plasmid also inhibits NSCLC tumor xenograft growth (16).
A growing list of genes have been identified that evidence abnormal CpG island promoter methylation (30). Methylation of the CpG island within the functional promoter region of WIF-1 is an important mechanism of aberrant Wnt signaling activation in cancer. Thus, by using methylation specific PCR and sequence analysis after bisulfite treatment, we demonstrated the frequent hypermethylation of the CpG islands in the functional WIF-1 promoter region, the hypermethylation correlated with their transcriptional silencing in human lung cancer cell lines (16). Conversely, promoter demethylation of WIF-1 restored the silenced WIF-1.
Procaine has been previously reported, in breast cancer cells, to cause global DNA hypomethylation, growth inhibition, and demethylation and re-expression of a methylation-silenced RAR-B2 (20). As such, procaine behaves very similarly to procainamide, the latter which specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1) (31), restores the expression of the hypermethylated GSTP1 gene in prostate cancer cells, and diminishes xenograft tumor growth (23). Therefore, we hypothesize that procaine and procainamide can also reactivate methylation-silenced WIF-1 gene.
In this study, we demonstrated that untreated H460 and A549 cells which were methylated silenced the expression of the WIF-1 gene. However, after exposure to procaine and procainamide, this epigenetic change was reversed. We also showed that H460 and A549 cells treated with procaine and procainamide decreased Tcf/Lef activity and expression of Bcatenin using a TOP/FOP Dual-Luciferase reporter assay and Western blot analysis, respectively.
These results are consistent with our hypothesis that procaine and procainamide may, in addition to restoring the methylated WIF-1 by demethylation, downregulate the Wnt canonical pathway in H460 and A549 cell lines.
The use of the nucleoside analogs such as DAC in clinical trials has been limited due to their side effects, including thrombocytopenia and neutropenia, probably caused by cytotoxic effects associated with the incorporation of the drugs into DNA, apart from their DNA-hypomethylation value.
There is thus an urgent need to discover less toxic demethylating agents that are not incorporated into DNA. Procaine and procainamide have been proposed as non-nucleosideinhibitors of DNA methylation. Their side effects are expected to be less as seen in nucleoside DNA methylation inhibitors because of their widespread clinical use (32).
We provided the first evidence that procaine and procainamide reactivate WIF-1 from a previously silenced methylation state and downregulate the canonical Wnt pathway. Procaine and procainmide may have a potential use for preventing the development of cancer.