The anti-proliferative effect of metformin in triple-negative MDA-MB-231 breast cancer cells is highly dependent on glucose concentration: Implications for cancer therapy and prevention

Background

Metformin has been shown to have a strong anti-proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP-activated protein kinase (AMPK). MDA-MB-231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti-cancer agents as well as to the anti-proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA-MB-231 cells to the anti-proliferative effect of metformin.

Methods

MDA-MB-231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by Western blotting.

Results

In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA-MB-231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA-MB-231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin-treatment of MDA-MB-231 cells cultured in normoglycemic conditions and not in hyperglycemic conditions caused a striking activation of AMPK, and an AMPK-dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation.

Conclusion

Our data show that normoglycemia sensitizes the triple negative MDA-MB-231 breast cancer cells to the anti-proliferative effect of metformin through an AMPK-dependent mechanism.

General significance

These findings suggest that tight normoglycemic control may enhance the anti-proliferative effect of metformin in diabetic cancer patients.     

Synthesis and in vitro anti-proliferative activity of some novel isatins conjugated with quinazoline/phthalazine hydrazines against triple-negative breast cancer MDA-MB-231 cells as apoptosis-inducing agents

Treatment of patients with triple-negative breast cancer (TNBC) is challenging due to the absence of well- defined molecular targets and the heterogeneity of such disease. In our endeavor to develop potent isatin-based anti-proliferative agents, we utilized the hybrid-pharmacophore approach to synthesize three series of novel isatin-based hybrids 5a–h, 10a–h and 13a–c, with the prime goal of developing potent anti-proliferative agents toward TNBC MDA-MB-231 cell line. In particular, compounds 5e and 10g were the most active hybrids against MDA-MB-231 cells (IC₅₀?=?12.35?±?0.12 and 12.00?±?0.13?μM), with 2.37- and 2.44-fold increased activity than 5-fluorouracil (5-FU) (IC₅₀?=?29.38?±?1.24?μM). Compounds 5e and 10g induced the intrinsic apoptotic mitochondrial pathway in MDA-MB-231; evidenced by the reduced expression of the anti-apoptotic protein Bcl-2, the enhanced expression of the pro-apoptotic protein Bax and the up-regulated active caspase-9 and caspase-3 levels. Furthermore, 10g showed significant increase in the percent of annexin V-FITC positive apoptotic cells from 3.88 to 31.21% (8.4 folds compared to control).     

Synthesis of novel isoflavene–propranolol hybrids as anti-tumor agents

Isoflavene–propranolol hybrid molecules were developed as potentially novel anti-tumour agents. Isoflavene itself has potent anti-cancer activity while propranolol can enhance anti-proliferative and anti-angiogenic properties of 5-fluorouracil and paclitaxel. The hybrids were produced via nucleophilic addition of substituted amine groups to a dioxiran intermediate, which was in turn generated from the Williamson-type reaction of isoflavene with (±)-epichlorohydrin. These analogues were tested in anti-cancer cell viability assays against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines, and were found to exhibit potent anti-proliferative activities. These compounds also displayed anti-angiogenic and anti-proliferative effects in HMEC-1 human microvascular endothelial cell lines. Notably, the most potent hybrid molecules synthesized in this work showed enhanced potency against cancer cell lines compared to either isoflavene or propranolol alone, while retaining significant selectivity for cancer cells over MRC-5 normal lung fibroblast cells.     

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.     

Natural inspired piperine-based ureas and amides as novel antitumor agents towards breast cancer

In this work, the natural piperine moiety was utilised to develop two sets of piperine-based amides (5a–i) and ureas (8a–y) as potential anticancer agents. The anticancer action was assessed against triple negative breast cancer (TNBC) MDA-MB-231, ovarian A2780CP and hepatocellular HepG2 cancer cell lines. In particular, 8q stood out as the most potent anti-proliferative analogue against TNBC MDA-MB-231 cells with IC₅₀ equals 18.7 µM, which is better than that of piperine (IC₅₀ = 47.8 µM) and 5-FU (IC₅₀ = 38.5 µM). Furthermore, 8q was investigated for its possible mechanism of action in MDA-MB-231 cells via Annexin V-FITC apoptosis assay and cell cycle analysis. Moreover, an in-silico analysis has proposed VEGFR-2 as a probable enzymatic target for piperine-based derivatives, and then has explored the binding interactions within VEGFR-2 active site (PDB:4ASD). Finally, an in vitro VEGFR-2 inhibition assay was performed to validate the in silico findings, where 8q showed good VEGFR-2 inhibitory activity with IC₅₀ = 231 nM.     

Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.     

Bystander killing of breast cancer MCF-7 cells by MDA-MB-231 cells exposed to 5-fluorouracil is mediated via Fas

The major drawback with cancer therapy is the development of resistant cells within tumors due to their heterogeneous nature and due to inadequate drug delivery during chemotherapy. Therefore, the propagation of injury ("bystander effect" (BE)) from directly damaged cells to other cells may have great implications in cancer chemotherapy. The general advantage of the bystander cell killing phenomenon is the large therapeutic index that can be achieved. Experiments suggest that this phenomenon is detected in radiation therapy as well as in gene therapy in conjunction with chemotherapy. In the present study, we developed an original in vitro model dedicated to the exploration of bystander cytotoxicity induced during breast carcinoma chemotherapy. In brief, we investigated this perpetuation of injury on untreated bystander MCF-7 breast cancer cells which were coplated with 5-fluorouracil (5-FU)-treated MDA-MB-231 breast cancer cells. To achieve this goal, a specific in vitro coculture model which involved mixing of aggressive MDA-MB-231 breast cancer cells with enhanced green fluorescent protein (EGFP) expressing stable clone of non-metastatic MCF-7 breast cancer cells (MCF-EGFP), was used. A bystander killing effect was observed in MCF-EGFP cells cocultured with MDA-MB-231 cells pretreated with 5-FU. The striking decrease in MCF-EGFP cells, as detected by assaying for total GFP intensity, is mediated by activation of Fas/FasL system. The implication of Fas in MCF-EGFP cell death was confirmed by using antagonistic anti-FasL antibody that reverses bystander cell death by blocking FasL on MDA-MB-231 cells. In addition, inhibition of CD95/Fas receptor on the cell surface of MCF-EGFP cells by treatment with Pifithrin-alpha, a p53 specific transactivation inhibitor, partially abrogated the sensitivity of bystander MCF-EGFP cells. Our data, therefore, demonstrates that the Fas/FasL system could be considered as a new determinant for chemotherapy-induced bystander cell death in breast cancers.     

Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase

Rac GTPases are known to play a crucial role in regulating cytoskeletal changes necessary for cell migration. Migration has been shown to be positively regulated by Rac in most cell types. However, there is also a large body of conflicting evidence in some other cell types with respect to the role of Rac in migration, suggesting that Rac GTPases regulate cell migration in a cell type-dependent manner. In the present study, we have characterized the effects of Rac1 GTPase inhibition on the migratory abilities of a number of breast cancer cell lines with differential degrees of tumorigenic and metastatic potentials. We show that Rac1 inhibition in non-metastatic (MCF-7, T-47D) or moderately metastatic (Hs578T) cell lines results in inhibition of migration, whereas in highly metastatic cell lines (MDA-MB-435, MDA-MB-231, and C3L5) Rac1 inhibition results in stimulation of migration. This stimulation of migration following Rac1 inhibition is also accompanied by the enhanced RhoA activity, suggesting a possible existence of a dominating role of RhoA over Rac1 in regulating intrinsic migration of the highly metastatic breast cancer cells.     

Synthesis of isoflavene-thiosemicarbazone hybrids and evaluation of their anti-tumor activity

Phenoxodiol is an isoflavene with potent anti-tumor activity. In this study, a series of novel mono- and di-substituted phenoxodiol-thiosemicarbazone hybrids were synthesized via the condensation reaction between phenoxodiol with thiosemicarbazides. The in vitro anti-proliferative activities of the hybrids were evaluated against the neuroblastoma SKN-BE(2)C, the triple negative breast cancer MDA-MB-231, and the glioblastoma U87 cancer cell lines. The mono-substituted hybrids exhibited potent anti-proliferative activity against all three cancer cell lines, while the di-substituted hybrids were less active. Selected mono-substituted hybrids were further investigated for their cytotoxicity against normal MRC-5 human lung fibroblast cells, which identified two hybrids with superior selectivity for cancer cells over normal cells as compared to phenoxodiol. This suggests that mono-substituted phenoxodiol-thiosemicarbazone hybrids have promising potential for further development as anti-cancer agents.     

IKK-β mediates chemoresistance by sequestering FOXO3; a critical factor for cell survival and death

Chemotherapeutic drugs proved only 50% successful in breast cancer because of cell type-dependent resistance mechanisms. FOXO3 is known to be involved in the regulation of several cell death-related genes; however, the extent of FOXO3 regulation in chemoresistance is still not fully understood. Here, we show that FOXO3 critically mediates cisplatin chemosensitivity of MCF-7 breast cancer cells which express higher levels of FOXO3 compared to resistant MDA-MB-231 cells. Administration of cisplatin induces apoptosis in MCF-7 cells in a FOXO3-dependent manner as indicated by RNA interference. On the other hand, IKK-β (IκB kinase) appears to inhibit FOXO3 action after cisplatin treatment and promotes chemoresistance in MDA-MB-231 cells. IKK-β directly interacts and sequesters FOXO3 in the cytosol preventing its nuclear localization. Moreover, cisplatin treatment induces autophagosome formation through LC-3 conversion while inhibiting the cleavage of caspase 9 and caspase 3 in MDA-MB-231 cells manipulated to overexpress FOXO3. In brief, our findings demonstrate that in addition to cellular level of active FOXO3, cisplatin chemoresistance is also regulated by IKK-β sequestration of FOXO3 in cytosol.     

Matrine suppresses breast cancer cell proliferation and invasion via VEGF-Akt-NF-<Emphasis Type="Italic">κ</Emphasis>B signaling

Matrine has shown therapeutic and/or adjuvant therapeutic effects on the treatment of some patients with breast cancer. However, its mechanisms of action are largely unknown. To disclose the mechanisms, we investigated in vitro and ex vivo effects of matrine on the cancer cells. Our results confirmed that matrine significantly suppressed the proliferation of highly-metastatic human breast cancer MDA-MB-231 cell line. Matrine displayed synergistic effects with existing anticancer agents celecoxib (the inhibitor of cyclooxygenase-2), trichostatin A (the histone deacetylase inhibitor) and rosiglitazone against the proliferation and VEGF excretions in MDA-MB-231 cells. Matrine induced the apoptosis and cell cycle arrest by reducing the ratios of Bcl-2/Bax protein and mRNA levels in the cancer cells. Matrine significantly reduced the invasion, MMP-9/MMP-2 activation, Akt phosphorylation, nuclear factor κB p-65 expression and DNA binding activity, and mRNA levels of MMP-9, MMP-2, EGF and VEGFR1 in MDA-MB-231 cells. Collectively, our results suggest that matrine inhibits the cancer cell proliferation and invasion via EGF/VEGF-VEGFR1-Akt-NF-κB signaling pathway.     

Artemisia absinthium (AA): a novel potential complementary and alternative medicine for breast cancer

Natural products have become increasingly important in pharmaceutical discoveries, and traditional herbalism has been a pioneering specialty in biomedical science. The search for effective plant-derived anticancer agents has continued to gain momentum in recent years. The present study aimed to investigate the role of crude extracts of the aerial parts of Artemisia absinthium (AA) extract in modulating intracellular signaling mechanisms, in particular its ability to inhibit cell proliferation and promote apoptosis in a human breast carcinoma estrogenic-unresponsive cell line, MDA-MB-231, and an estrogenic-responsive cell line, MCF-7. Cells were incubated with various concentrations of AA, and anti-proliferative activity was assessed by MTT assays, fluorescence microscopy after propidium iodide staining, western blotting and cell cycle analysis. Cell survival assays indicated that AA was cytotoxic to both MDA-MB-231 and MCF-7 cells. The morphological features typical of nucleic staining and the accumulation of sub-G1 peak revealed that the extract triggered apoptosis. Treatment with 25 μg/mL AA resulted in activation of caspase-7 and upregulation of Bad in MCF-7 cells, while exposure to 20 μg/mL AA induced upregulation of Bcl-2 protein in a time-dependent response in MDA-MB-231 cells. Both MEK1/2 and ERK1/2 was inactivated in both cell lines after AA treatment in a time-dependent manner. These results suggest that AA-induced anti-proliferative effects on human breast cancer cells could possibly trigger apoptosis in both cell lines through the modulation of Bcl-2 family proteins and the MEK/ERK pathway. This might lead to its possible development as a therapeutic agent for breast cancer following further investigations.     

Potential anti-proliferative agents from 1,4-benzoxazinone-quinazolin-4(3H)-one templates

A novel synthetic protocol has been developed for the synthesis of 1,4-benzoxazinone-acetylphenylallyl quinazolin-4(3H)-one hybrids 7a–n by employing Pd-catalyzed CH arylation in presence of 5–10% phosphine ligand in good to excellent yields and evaluated for their anti-proliferative activity against three cancer cell lines such as A549 (lung), HeLa (cervical), MDA-MB-231 (breast). Compounds 7d, 7f, 7l and 7n exhibited promising anti-proliferative activity with GI₅₀ values ranging from 0.37 to 2.73?µM respectively against A549, HeLa, and MDA-MB-231, while compound 7f showed significant activity against MDA-MB-231 with GI₅₀ value 0.58?µM, 7j showed significant activity against A549 with GI₅₀ value 0.32?µM and 7l showed significant activity against HeLa with GI₅₀ value 0.37?µM. This is the first report on the synthesis and in vitro anti-proliferative evaluation of 1,4-benzoxazinone-acetylphenylallyl quinazolin-4(3H)-one hybrids.     

Anandamide inhibits Cdk2 and activates Chk1 leading to cell cycle arrest in human breast cancer cells

This study was designed to determine the molecular mechanisms underlying the anti-proliferative effect of the endocannabinoid anandamide on highly invasive human breast cancer cells, MDA-MB-231. We show that a metabolically stable analogue of anandamide, Met-F-AEA, induces an S phase growth arrest correlated with Chk1 activation, Cdc25A degradation and suppression of Cdk2 activity. These findings demonstrate that Met-F-AEA induced cell cycle blockade relies on modulated expression and activity of key S phase regulatory proteins. The observed mechanism of action, already reported for well-known chemotherapeutic drugs, provides strong evidence for a direct role of anandamide related compounds in the activation of cell cycle checkpoints.     

二氢杨梅素抑制人乳腺癌细胞侵袭和下调MMP-2/-9蛋白表达研究

藤茶活性成分二氢杨梅素(3, 5, 7, 3′, 4′, 5′-六羟基-2, 3-二氢黄酮醇,DMY)体外对几种癌细胞具有抗增殖作用,但机制尚未完全清楚．本文研究DMY对人高转移型乳腺癌MDA-MB-231细胞侵袭的影响,并探讨可能的机制．用MTT法检测DMY对MDA-MB-231细胞的增殖抑制率;明胶酶谱分析明胶酶活力;基质金属蛋白酶(MMP-2/-9)的基因表达水平和蛋白质表达水平分别利用实时定量PCR和Western blot分析进行检测．Transwell模型检测DMY对肿瘤细胞侵袭的影响．结果显示,DMY以剂量依赖方式抑制MDA-MB-231细胞的增殖,作用48 h的IC50为73.6 mg/L．DMY显著抑制明胶酶活性和MMP-2/-9蛋白表达,并抑制MMP-2/-9 的mRNA表达水平．此外,DMY不依赖细胞毒作用和以剂量依赖方式抑制MDA- MB-231细胞的侵袭．这些结果提示：DMY能显著抑制人乳腺癌MDA-MB-231细胞的侵袭和增殖, 其侵袭抑制的机制可能与其下调MMP-2/-9蛋白表达水平相关．     

Metformin reverses multidrug resistance and epithelial–mesenchymal transition (EMT) via activating AMP-activated protein kinase (AMPK) in human breast cancer cells

Breast cancer is the most frequently diagnosed tumor type and the primary leading cause of cancer deaths in women worldwide and multidrug resistance is the major obstacle for breast cancer treatment improvement. Emerging evidence suggests that metformin, the most widely used antidiabetic drug, resensitizes and cooperates with some anticancer drugs to exert anticancer effect. However, there are no data regarding the reversal effect of metformin on chemoresistance in breast cancer. In the present study, we investigated the resistance reversal effect of metformin on acquired multidrug-resistant breast cancer cells MCF-7/5-Fu derived from MCF-7 breast cancer cells and innate multidrug-resistant MDA-MB-231 breast cancer cells, and we found that metformin resensitized MCF7/5-FU and MDA-MB-231 to 5-fluorouracil (5-FU), adriamycin, and paclitaxel. We also observed that metformin reversed epithelial–mesenchymal transition (EMT) phenotype and decreased the invasive capacity of MCF7/5-FU and MDA-MB-231 cells. However, there were no significant changes upon metformin-treated MCF7 cells. Moreover, we found metformin treatment activated AMPK signal pathway in MCF7/5-FU and MDA-MB-231 cells and compound C, the AMPK inhibitor, could partly abolish the resensitization and EMT reversal effect of metformin. To the best of our knowledge, we are the first to report that metformin can resensitize multidrug-resistant breast cancer cells due to activating AMPK signal pathway. Our study will help elucidate the mechanism of chemoresistance and establish new strategies of chemotherapy for human breast cancer.     

Synthesis, characterization and anti-tumor activity of moxifloxacin-copper complexes against breast cancer cell lines

Novel moxifloxacin-copper complexes were synthesized, characterized and screened for anti-proliferative and apoptosis-inducing activity against multiple human breast cancer cell lines (hormone-dependent MCF-7 and T47D as well as hormone-independent MDA-MB-231 and BT-20). The results indicated that the parent compound moxifloxacin (1) does not exert any inhibitory activity against breast cancer cell lines examined. On the other hand, the copper conjugate 2 and its nitrogen adducts 3-5 exerted growth inhibitory and apoptosis-inducing activity against breast cancer cell lines without any substantial effect on non-tumorigenic breast epithelial cells MCF-10A at equimolar concentration, suggesting a cancer cell-specific activity. BT-20 cells were more sensitive to compounds 2 and 3, while compounds 4 and 5 exerted significant anti-proliferative and apoptosis-inducing effects on T47D, MDA-MB-231 and BT-20 cell lines. Our results suggest that these novel compounds could be useful for the treatment of breast cancer in the future.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

Molecular characterization of a dual inhibitory and mutagenic activity of 5-fluorouridine triphosphate on viral RNA synthesis. Implications for lethal mutagenesis

The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-fluorouridine triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.