Antisense inhibition of methylenetetrahydrofolate reductase reduces survival of methionine-dependent tumour lines

Abstract

Transformed cells have been documented to be methionine-dependent, suggesting that inhibition of methionine synthesis might be useful for cancer therapy. Methylenetetrahydrofolate reductase synthesises 5-methyltetrahydrofolate, the methyl donor utilised in methionine synthesis from homocysteine by vitamin B 12 -dependent methionine synthase. We hypothesised that methylenetetrahydrofolate reductase inhibition would affect cell viability through decreased methionine synthesis. Using medium lacking methionine, but containing homocysteine and vitamin B 12 (M-H+), we found that nontransformed human fibroblasts could maintain growth. In contrast, four transformed cell lines (one colon carcinoma, two neuroblastoma and one breast carcinoma) increased proliferation only slightly in the M-H+ medium. To downregulate methylenetetrahydrofolate reductase expression, two phosphorothioate antisense oligonucleotides, EX5 and 677T, were used to target methylenetetrahydrofolate reductase in the colon carcinoma line SW620; 400 nM of each antisense oligonucleotide decreased cell survival by approximately 80% (P50.01) and 70% (P50.0001), respectively, compared to cell survival after the respective control mismatched oligonucleotide. Western blotting and enzyme assays confirmed that methylenetetrahydrofolate reductase expression was decreased. Two neuroblastoma and two breast carcinoma lines also demonstrated decreased survival following EX5 treatment whereas nontransformed human fibroblasts were not affected. This study suggests that methylenetetrahydrofolate reductase may be required for tumour cell survival and that methylenetetrahydrofolate reductase inhibition should be considered for anti-tumour therapy.

Expression Profiling of Homocysteine Junction Enzymes in the NCI60 Panel of Human Cancer Cell Lines

“…These include A498 (renal), PC-3 (prostate), MCF7 (breast), SW-620 (colon), HL-60 (leukemia), K-562 (leukemia), and A549 (non-small cell lung carcinoma; refs. 3,27,28,30). MS and MSR were expressed in all seven of these cell lines at levels that were comparable to those in other cell lines originating from the same tissue.…”

Antisense Inhibition of Methylenetetrahydrofolate Reductase Reduces Cancer Cell Survival In vitro and Tumor Growth In vivo

“…Previous studies in our laboratory with MTHFR antisense resulted in significant reductions of MTHFR protein that affected survival of methionine-dependent cancer cell lines, whereas nontransformed cells (human diploid fibroblasts) were not affected. EX5 of the human MTHFR mRNA was determined to be quite effective and examined in this study, although an antisense against exon 4 also had growth inhibitory properties (12).…”

“…The EX5 MTHFR specific antisense (EX5; sequence 5V -AGC TGC CGA AGG GAG TGG TA-3V) targets nucleotides 796 to 815 in exon 5 of the human MTHFR mRNA. Selection of EX5 was based on the results of our previous work (12). The control scrambled antisense oligodeoxynucleotide (CTS; sequence 5V -TGT TGA CAA AAG GAG TGG TG-3V) has the same base composition in random order.…”

“…Western blotting for MTHFR and actin was done as previously described (12). Quantification of protein was determined by scanning the films with a flatbed scanner (Hewlett Packard Scan, Palo Alto, CA).…”

Gene therapy for carcinoma of the breast

“…51 In addition to c-myc, 52 other genes relevant to breast cancer have been successfully targeted by antisense oligonucleotides such as aV integrin, 53 ribosomal protein P2, 54 c-erbB-2, 55 methylenetetrahydrofolate reductase (MTHFR), 56 p21, 57 protein kinase C-alpha (PKC-a), 58,59 Bcl-2, 60 telomerase reverse transcriptase and telomerase, 61 plasma membrane calcium ATPase, 62 type I insulin-like growth factor receptor (IGF-IR), 63 and c-fos. 52 Compared with preclinical studies, far fewer clinical studies of oligonucleotides have been reported.…”