REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint.

The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3-/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3-/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3-/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3-/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance.

Floxuridine supports UPS independent of germline signaling and proteostasis regulators via involvement of detoxification in C. elegans.

The ubiquitin-proteasome system (UPS) is critical for maintaining proteostasis, influencing stress resilience, lifespan, and thermal adaptability in organisms. In Caenorhabditis elegans, specific proteasome subunits and activators, such as RPN-6, PBS-6, and PSME-3, are associated with heat resistance, survival at cold (4°C), and enhanced longevity at moderate temperatures (15°C). Previously linked to improving proteostasis, we investigated the impact of sterility-inducing floxuridine (FUdR) on UPS functionality under proteasome dysfunction and its potential to improve cold survival. Our findings reveal that FUdR significantly enhances UPS activity and resilience during proteasome inhibition or subunit deficiency, supporting worms' normal lifespan and adaptation to cold. Importantly, FUdR effect on UPS activity occurs independently of major proteostasis regulators and does not rely on the germ cells proliferation or spermatogenesis. Instead, FUdR activates a distinct detoxification pathway that supports UPS function, with GST-24 appearing to be one of the factors contributing to the enhanced activity of the UPS upon knockdown of the SKN-1-mediated proteasome surveillance pathway. Our study highlights FUdR unique role in the UPS modulation and its crucial contribution to enhancing survival under low-temperature stress, providing new insights into its mechanisms of action and potential therapeutic applications.

High-load nanoparticles with a chemotherapeutic SN-38/FdUMP drug cocktail.

[Gd(OH)]2+[(SN-38)0.5(FdUMP)0.5]2- inorganic-organic hybrid nanoparticles (IOH-NPs) with a chemotherapeutic cocktail of ethyl-10-hydroxycamptothecin (SN-38, active form of irinotecan) and 5-fluoro-2'-deoxyuridine-5'-phosphate (FdUMP, active form of 5'-fluoruracil), 40 nm in size, are prepared in water. The IOH-NPs contain a total drug load of 63 wt% with 33 wt% of SN-38 and 30 wt% of FdUMP. Cell-based assays show efficient cellular uptake and promising anti-tumour activity on two pancreatic cancer cell lines of murine origin (KPC, Panc02). Beside the high-load drug cocktail, especially the option to use SN-38, which - although 100- to 1000-times more potent than irinotecan - is usually unsuitable for systemic administration due to poor solubility, low stability, and high toxicity upon non-selective delivery. The [Gd(OH)]2+[(SN-38)0.5(FdUMP)0.5]2- IOH-NPs are a new concept to deliver a drug cocktail with SN-38 and FdUMP directly to the tumour, shielded in a nanoparticle, to reduce side effects.

Development of a Supermolecular Radionuclide-Drug Conjugate System for Integrated Radiotheranostics for Non-small Cell Lung Cancer.

Radionuclide-drug conjugates (RDCs) designed from small molecule or nanoplatform shows complementary characteristics. We constructed a new RDC system with integrated merits of small molecule and nanoplatform-based RDCs. Erlotinib was labeled with 131I to construct the bulk of RDC (131I-ER). Floxuridine was mixed with 131I-ER to develop a hydrogen bond-driving supermolecular RDC system (131I-ER-Fu NPs). The carrier-free 131I-ER-Fu NPs supermolecule not only demonstrated integrated merits of small molecule and nanoplatform-based RDC, including clear structure definition, stable quality control, prolonged circulation lifetime, enhanced tumor specificity and retention, and rapidly nontarget clearance, but also exhibited low biological toxicity and stronger antitumor effects. In vivo imaging also revealed its application for tumor localization of nonsmall cell lung cancer (NSCLC) and screening of patients suitable for epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy. We considered that 131I-ER-Fu NPs showed potentials as an integrated platform for the radiotheranostics of NSCLC.

Case-Based Clinical Guidance on Dosing and Management of the Hepatic Artery Infusion Chemotherapy Pump.

Hepatic artery infusion (HAI) delivers localized high-dose floxuridine directly to liver tumors through an implanted pump. While patients are undergoing active treatment, the pump is refilled with chemotherapy alternating with saline every 2 weeks using a specialized noncoring needle. Numerous clinical scenarios influence the dosing of floxuridine, which do not conform to the usual dose modification schema for systemic chemotherapy. This article aims to provide practical clinical management solutions to overcome the common challenges faced by oncologists in the real-world management of HAI pump therapy.

Effects of six pyrimidine analogs on the growth of Tetrahymena thermophila and their implications in pyrimidine metabolism.

Tetrahymena are ciliated protists that have been used to study the effects of toxic chemicals, including anticancer drugs. In this study, we tested the inhibitory effects of six pyrimidine analogs (5-fluorouracil, floxuridine, 5'-deoxy-5-fluorouridine, 5-fluorouridine, gemcitabine, and cytarabine) on wild-type CU428 and conditional mutant NP1 Tetrahymena thermophila at room temperature and the restrictive temperature (37°C) where NP1 does not form the oral apparatus. We found that phagocytosis was not required for pyrimidine analog entry and that all tested pyrimidine analogs inhibited growth except for cytarabine. IC50 values did not significantly differ between CU428 and NP1 for the same analog at either room temperature or 37°C. To investigate the mechanism of inhibition, we used two pyrimidine bases (uracil and thymine) and three nucleosides (uridine, thymidine, and 5-methyluridine) to determine whether the inhibitory effects from the pyrimidine analogs were reversible. We found that the inhibitory effects from 5-fluorouracil could be reversed by uracil and thymine, from floxuridine could be reversed by thymidine, and from 5'-deoxy-5-fluorouridine could be reversed by uracil. None of the tested nucleobases or nucleosides could reverse the inhibitory effects of gemcitabine or 5-fluorouridine. Our results suggest that the five pyrimidine analogs act on different sites to inhibit T. thermophila growth and that nucleobases and nucleosides are metabolized differently in Tetrahymena.

Adjusting the neuron to astrocyte ratio with cytostatics in hippocampal cell cultures from postnatal rats: A comparison of cytarabino furanoside (AraC) and 5-fluoro-2'-deoxyuridine (FUdR).

Cell culture studies offer the unique possibility to investigate the influence of pharmacological treatments with quantified dosages applied for defined time durations on survival, morphological maturation, protein expression and function as well as the mutual interaction of various cell types. Cultures obtained from postnatal rat brain contain a substantial number of glial cells that further proliferate with time in culture leading to an overgrowth of neurons with glia, especially astrocytes and microglia. A well-established method to decrease glial proliferation in vitro is to apply low concentrations of cytosine arabinoside (AraC). While AraC primarily effects dividing cells, it has been reported repeatedly that it is also neurotoxic, which is the reason why most protocols limit its application to concentrations of up to 5 µM for a duration of 24 h. Here, we investigated 5-fluoro-2'-deoxyuridine (FUdR) as a possible substitute for AraC. We applied concentrations of both cytostatics ranging from 4 µM to 75 µM and compared cell composition and cell viability in cultures prepared from 0-2- and 3-4-day old rat pups. Using FUdR as proliferation inhibitor, higher ratios of neurons to glia cells were obtained with a maximal neuron to astrocyte ratio of up to 10:1, which could not be obtained using AraC in postnatal cultures. Patch-clamp recordings revealed no difference in the amplitudes of voltage-gated Na+ currents in neurons treated with FUdR compared with untreated control cells suggesting replacement of AraC by FUdR as glia proliferation inhibitor if highly neuron-enriched postnatal cultures are desired.

DNA-Damage-Response-Targeting Mitochondria-Activated Multifunctional Prodrug Strategy for Self-Defensive Tumor Therapy.

We report a novel multifunctional construct, M1, designed explicitly to target the DNA damage response in cancer cells. M1 contains both a floxuridine (FUDR) and protein phosphatase 2A (PP2A) inhibitor combined with a GSH-sensitive linker. Further conjugation of the triphenylphosphonium moiety allows M1 to undergo specific activation in the mitochondria, where mitochondria-mediated apoptosis is observed. Moreover, M1 has enormous effects on genomic DNA ascribed to FUDR's primary function of impeding DNA/RNA synthesis combined with diminishing PP2A-activated DNA repair pathways. Importantly, mechanistic studies highlight the PP2A obtrusion in FUDR/5-fluorouracil (5-FU) therapy and underscore the importance of its inhibition to harbor therapeutic potential. HCT116 cell xenograft-bearing mice that have a low response rate to 5-FU show a prominent effect with M1, emphasizing the importance of DNA damage response targeting strategies using tumor-specific microenvironment-activatable systems.

Aptamers Entirely Built from Therapeutic Nucleoside Analogues for Targeted Cancer Therapy.

Owing to the specific and high binding affinity of aptamers to their targets, aptamer-drug conjugates (ApDCs) have emerged as a promising drug delivery system for targeted cancer therapy. However, in a conventional ApDC, the aptamer segment usually just serves as a targeting moiety, and only a limited number of drug molecules are sequentially conjugated to the oligonucleotide, giving a relatively low drug loading capacity. To address this challenge, herein we employ four clinically approved nucleoside analogues, including clofarabine (Clo), ara-guanosine (AraG), gemcitabine (Ge), and floxuridine (FdU), to replace all natural nucleosides in aptamer sequences, generating a series of whole drug-constituted DNA-like oligomers that are termed drugtamers. Similar to their parent aptamers, the obtained drugtamers maintain the targeting capability and can specifically bind to the target receptors overexpressed on the cancer cell surface. With 100% drug loading ratio, active targeting capability, and enzyme-mediated release of active therapeutics, our drugtamers can strongly induce the apoptosis of cancer cells and inhibit the tumor progression, which enables a new potential for a better targeted cancer therapy.

Construction of synergistic pH/H2O2-responsive prodrug for prolonging blood circulation and accelerating cellular internalization.

We have developed a real-time and multifunctional doxifluridine-conjugate prodrug (LYX), which involved the preliminary methylfluorescein with 5-fluorouracil linker as protecting group, the targeting biotin unit, and a model therapeutic drug (doxifluridine). The shielding group (5'-DFUR) was found to be effective in prolonging circulation at physiological pH 7.4 and improving accumulation in the acidic microenvironment of the tumor. Based on this strategy, the stability and stimulus responsive properties of prodrug could enhance drug release efficiency and exhibit fewer side effects, thereby providing a unique opportunity for diagnosis and imaging additional analytes or enzymatic activities.

Evaluation of Floxuridine Oligonucleotide Conjugates Carrying Potential Enhancers of Cellular Uptake.

Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been used to improve their pharmacological properties. In this work, we studied the biological effects of several small-molecule enhancers into a short oligonucleotide made of five floxuridine units. Specifically, we studied adding cholesterol, palmitic acid, polyethyleneglycol (PEG 1000), folic acid and triantennary N-acetylgalactosamine (GalNAc) as potential enhancers of cellular uptake. As expected, all these molecules increased the internalization efficiency with different degrees depending on the cell line. The conjugates showed antiproliferative activity due to their metabolic activation by nuclease degradation generating floxuridine monophosphate. The cytotoxicity and apoptosis assays showed an increase in the anti-cancer activity of the conjugates related to the floxuridine oligomer, but this effect did not correlate with the internalization results. Palmitic and folic acid conjugates provide the highest antiproliferative activity without having the highest internalization results. On the contrary, cholesterol oligomers that were the best-internalized oligomers had poor antiproliferative activity, even worse than the unmodified floxuridine oligomer. Especially relevant is the effect induced by palmitic and folic acid derivatives generating the most active drugs. These results are of special interest for delivering other therapeutic oligonucleotides.

Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells.

5-Fluorouracil (5-FU) is a cornerstone drug used in the treatment of colorectal cancer (CRC). However, the development of resistance to 5-FU and its analogs remain an unsolved problem in CRC treatment. In this study, we investigated the molecular mechanisms and tumor biological aspects of 5-FU resistance in CRC HCT116 cells. We established an acquired 5-FU-resistant cell line, HCT116RF10. HCT116RF10 cells were cross-resistant to the 5-FU analog, fluorodeoxyuridine. In contrast, HCT116RF10 cells were collaterally sensitive to SN-38 and CDDP compared with the parental HCT16 cells. Whole-exome sequencing revealed that a cluster of genes associated with the 5-FU metabolic pathway were not significantly mutated in HCT116 or HCT116RF10 cells. Interestingly, HCT116RF10 cells were regulated by the function of thymidylate synthase (TS), a 5-FU active metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) inhibiting enzyme. Half of the TS was in an active form, whereas the other half was in an inactive form. This finding indicates that 5-FU-resistant cells exhibited increased TS expression, and the TS enzyme is used to trap FdUMP, resulting in resistance to 5-FU and its analogs.

Inhibition of Human Uracil DNA Glycosylase Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-Fluorodeoxyuridine and Raltitrexed but Not Fluorouracil.

Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.

Mechanism of acquired 5FU resistance and strategy for overcoming 5FU resistance focusing on 5FU metabolism in colon cancer cell lines.

Fluorouracil (5FU) is converted to its active metabolite fluoro­deoxyuridine monophosphate (FdUMP) through the orotate phosphoribosyl transferase (OPRT)­ribonucleotide reductase (RR) pathway and thymidine phosphatase (TP)­thymidine kinase (TK) pathway and inhibits thymidylate synthase (TS), leading to inhibition of thymidine monophosphate (dTMP) synthesis through a de novo pathway. We investigated the mechanism of 5FU resistance and strategies to overcome it by focusing on 5FU metabolism. Colon cancer cell lines SW48 and LS174T and 5FU­resistant cell lines SW48/5FUR and LS174T/5FUR were used. FdUMP amount was measured by western blotting. The FdUMP synthetic pathway was investigated by combining TP inhibitor (tipiracil hydrochloride; TPI) or RR inhibitor (hydroxyurea; HU) with 5FU. Drug cytotoxicity was observed by crystal violet staining assay. FdUMP was synthesized through the OPRT­RR pathway in SW48 cells but was scarcely synthesized through either the OPRT­RR or TP­TK pathway in SW48/5FUR cells. FdUMP amount in SW48/5FUR cells was reduced by 87% vs. SW48 cells. Expression levels of OPRT and TP were lower in SW48/5FUR when compared with these levels in the SW48 cells, indicating decreased synthesis of FdUMP­led 5FU resistance. These results indicated that fluoro­deoxyuridine (FdU) rather than 5FU promotes FdUMP synthesis and overcomes 5FU resistance. Contrastingly, FdUMP was synthesized through the OPRT­RR and TP­TK pathways in LS174T cells but mainly through the TP­TK pathway in LS174T/5FUR cells. FdUMP amount was similar in LS174T/5FUR vs. the LS174T cells. OPRT and RR expression was lower and TK expression was higher in LS174T/5FUR vs. the LS174T cells, indicating that dTMP synthesis increased through the salvage pathway, thus leading to 5FU resistance. LS174T/5FUR cells also showed cross­resistance to FdU and TS inhibitor, suggesting that nucleoside analogs such as trifluoro­thymidine should be used to overcome 5FU resistance in these cells. 5FU metabolism and mechanisms of 5FU resistance are different in each cell line. Both synthesized FdUMP amount and FdUMP sensitivity should be considered in 5FU­resistant cells.

The selective serotonin reuptake inhibitors enhance the cytotoxicity of sorafenib in hepatocellular carcinoma cells.

Sertraline and fluoxetine are the two most commonly used selective serotonin reuptake inhibitors (SSRIs) in the treatment of depression. Accumulating evidence has revealed that SSRIs can reduce the risk of hepatocellular carcinoma (HCC), but their therapeutic effects in HCC have not yet been elucidated. Previous studies have reported that sertraline and fluoxetine can suppress the growth of gastric carcinoma, melanoma and nonsmall cell lung cancers by inhibiting the mammalian target rapamycin (mTOR) activity. In this study, we found that sertraline and fluoxetine blocked the protein kinase B (AKT)/mTOR pathway and suppressed the growth of HCC cells in vitro, in xenografts and in diethylnitrosamine/carbon tetrachloride (DEN/CCL4)-induced primary liver mouse model. Sertraline and fluoxetine can synergize with sorafenib, the first approved standard therapy for advanced HCC, to inhibit the viability of HCC cells in vitro and in vivo. In addition, the combination of sorafenib and SSRIs synergistically inhibited the effects of the AKT/mTOR pathway. These results reveal novel therapeutic effects of a combination of SSRIs and sorafenib in HCC.

dUTPase inhibition confers susceptibility to a thymidylate synthase inhibitor in DNA-repair-defective human cancer cells.

Deficiency in DNA repair proteins confers susceptibility to DNA damage, making cancer cells vulnerable to various cancer chemotherapies. 5-Fluorouracil (5-FU) is an anticancer nucleoside analog that both inhibits thymidylate synthase (TS) and causes DNA damage via the misincorporation of FdUTP and dUTP into DNA under the conditions of dTTP depletion. However, the role of the DNA damage response to its antitumor activity is still unclear. To determine which DNA repair pathway contributes to DNA damage caused by 5-FU and uracil misincorporation, we examined cancer cells treated with 2'-deoxy-5-fluorouridine (FdUrd) in the presence of TAS-114, a highly potent inhibitor of dUTPase that restricts aberrant base misincorporation. Addition of TAS-114 increased FdUTP and dUTP levels in HeLa cells and facilitated 5-FU and uracil misincorporation into DNA, but did not alter TS inhibition or 5-FU incorporation into RNA. TAS-114 showed synergistic potentiation of FdUrd cytotoxicity and caused aberrant base misincorporation, leading to DNA damage and induced cell death even after short-term exposure to FdUrd. Base excision repair (BER) and homologous recombination (HR) were found to be involved in the DNA repair of 5-FU and uracil misincorporation caused by dUTPase inhibition in genetically modified chicken DT40 cell lines and siRNA-treated HeLa cells. These results suggested that BER and HR are major pathways that protect cells from the antitumor effects of massive incorporation of 5-FU and uracil. Further, dUTPase inhibition has the potential to maximize the antitumor activity of fluoropyrimidines in cancers that are defective in BER or HR.

Co-delivery of 5-fluorodeoxyuridine and doxorubicin via gold nanoparticle equipped with affibody-DNA hybrid strands for targeted synergistic chemotherapy of HER2 overexpressing breast cancer.

Combination chemotherapy is still of great importance as part of the standard clinical care for patients with HER2 positive breast cancer. As an attractive component, gold nanoparticles (AuNPs) have been extensively studied as biosafety nanomaterials, but they are rarely explored as drug nanocarriers for targeted co-delivery of multiple chemotherapeutics. Herein, a novel affibody-DNA hybrid strands modified AuNPs were fabricated for co-loading nucleoside analogue (5-fluorodeoxyuridine, FUdR) and anthracycline (doxorubicin, Dox). FUdRs were integrated into DNA hybrid strands decorated on AuNPs by DNA solid phase synthesis, and Dox molecules were intercalated into their duplex regions. Affibody molecules coupled to the DNA hybrid strands were distributed the surface of AuNPs, giving them targeting for HER2. The new dual-drug-containing affibody-DNA-AuNPs (Dox@affi-F/AuNPs) owned compact and stable spherical nanostructures, and precise drug loading. Cytotoxicity tests demonstrated that these nanoparticles caused a higher inhibition in HER2 overexpressing breast cancer cells, and showed better synergistic antitumor activity than simple mixture of the two drugs. The related mechanistic studies proved that Dox@affi-F/AuNPs achieved a remarkable combined antitumor activity of Dox and FUdR by promoting more cells to enter apoptosis pathway. Our work provided a nanomedicine platform for targeted co-delivery of nucleoside analog therapeutics and anthracycline anticancer drugs to achieve synergistic treatment of HER2+ cancer.

Evolved bacterial resistance against fluoropyrimidines can lower chemotherapy impact in the Caenorhabditis elegans host.

Metabolism of host-targeted drugs by the microbiome can substantially impact host treatment success. However, since many host-targeted drugs inadvertently hamper microbiome growth, repeated drug administration can lead to microbiome evolutionary adaptation. We tested if evolved bacterial resistance against host-targeted drugs alters their drug metabolism and impacts host treatment success. We used a model system of Caenorhabditis elegans, its bacterial diet, and two fluoropyrimidine chemotherapies. Genetic screens revealed that most of loss-of-function resistance mutations in Escherichia coli also reduced drug toxicity in the host. We found that resistance rapidly emerged in E. coli under natural selection and converged to a handful of resistance mechanisms. Surprisingly, we discovered that nutrient availability during bacterial evolution dictated the dietary effect on the host - only bacteria evolving in nutrient-poor media reduced host drug toxicity. Our work suggests that bacteria can rapidly adapt to host-targeted drugs and by doing so may also impact the host.

Intracellular microRNA expression patterns influence cell death fates for both necrosis and apoptosis.

MicroRNAs (miRNAs) are small noncoding RNA molecules that interact with target mRNAs at specific sites to induce cleavage of the mRNA or inhibit translation. Such miRNAs play a vital role in gene expression and in several other biological processes, including cell death. We have studied the mechanisms regulating cell death (necrosis in original F28-7 cells and apoptosis in their variant F28-7-A cells) in the mouse mammary tumor cell line FM3A using the anticancer agent floxuridine (FUdR). We previously reported that inhibition of heat-shock protein 90 by the specific inhibitor geldanamycin (GA) in F28-7 cells causes a shift from necrosis to apoptosis. In this study, we investigated the intracellular miRNA expression profiles of FUdR-treated F28-7 cells (necrotic condition), GA plus FUdR-treated F28-7 cells (apoptotic condition), and FUdR-treated F28-7-A cells (apoptotic condition) through miRNA microarray analysis. In addition, we knocked down Dicer, a key molecule for the expression of mature miRNAs, in F28-7 cells to examine whether it modulates FUdR-induced cell death. Our analysis revealed that the miRNA expression patterns differ significantly between these cell death conditions. Furthermore, we identified miRNA candidates that regulate cell death. Knockdown of Dicer in FUdR-treated necrosis-fated cells caused a partial shift from necrosis to apoptosis. These findings suggest that modulation of miRNA expression patterns influences the decision of cell death fate toward necrosis or apoptosis. Our findings may serve as a basis for further study of the functions of miRNAs in cell death mechanisms.

Anticancer Strategy Targeting Cell Death Regulators: Switching the Mechanism of Anticancer Floxuridine-Induced Cell Death from Necrosis to Apoptosis.

Cell death can be broadly characterized as either necrosis or apoptosis, depending on the morphological and biochemical features of the cell itself. We have previously reported that the treatment of mouse mammary carcinoma FM3A cells with the anticancer drug floxuridine (FUdR) induces necrosis in the original clone F28-7 but apoptosis in the variant F28-7-A. We have identified regulators, including heat shock protein 90, lamin-B1, cytokeratin-19, and activating transcription factor 3, of cell death mechanisms by using comprehensive gene and protein expression analyses and a phenotype-screening approach. We also observed that the individual inhibition or knockdown of the identified regulators in F28-7 results in a shift from necrotic to apoptotic morphology. Furthermore, we investigated microRNA (miRNA, miR) expression profiles in sister cell strains F28-7 and F28-7-A using miRNA microarray analyses. We found that several unique miRNAs, miR-351-5p and miR-743a-3p, were expressed at higher levels in F28-7-A than in F28-7. Higher expression of these miRNAs in F28-7 induced by transfecting miR mimics resulted in a switch in the mode of cell death from necrosis to apoptosis. Our findings suggest that the identified cell death regulators may play key roles in the decision of cell death mechanism: necrosis or apoptosis.