Targeting Thymidylate Synthase Research Articles

Synthesis and Anticancer Evaluation of Benzylated Indole Based Alkyl‐Substituted Pyrido[2,3‐d]Pyrimidines Targeting Thymidylate Synthase

AbstractColorectal cancer (CRC) is the second most common cause of cancer‐related deaths and the third most common cancer worldwide. Because of its importance in DNA biosynthesis, thymidylate synthase (TS) has gathered attention as a promising target for cancer therapy. Herein, we have designed and synthesized 12 novel 5‐(N‐Benzyl indol‐3‐yl) pyrido[2, 3‐d] pyrimidine based derivatives and assessed their anticancer activity against HCT 116, MCF‐7, Hep G2, and PC‐3 cell lines. For compounds 5 a and 5 f TS inhibitory activity was also performed along with in silico studies. Further, all the synthesized compounds exhibited anticancer activity, but particularly, compounds 5 a and 5 f were showed excellent anticancer activity having IC50 values of 1.09±0.61, 5.54±0.81, 6.44±0.91, and 5.12±0.65 μM and 1.03±0.96, 3.09±0.52, 4.12±0.59, and 7.06±0.60 μM respectively with control Raltitrexed (IC50 1.02±0.51, 1.88±0.61, 1.30±0.89, and 2.09±0.67 μM respectively) and hTS inhibitory activity with IC50 of 18.91±1.09 and 16.19±0.96 nM with control Raltitrexed (IC50 12.25±1.21 nM). Further, the mechanism of inhibition was revealed by molecular docking, which showed the binding pattern of 5 a and 5 f to the catalytic site of TS. Additionally, ADME characteristics were also in the acceptable range for these compounds. Based on the anticancer activity, SAR was also performed for lead optimization.

An insight into thymidylate synthase inhibitor as anticancer agents: an explicative review.

Cancer, a widespread challenge to global health, remains a puzzle of intricate molecular dynamics. This review article delves into the mystery of cancer, with a keen focus on understanding the contributory role of thymidylate synthase (TS) in cancer. TS, a vital enzyme in DNA synthesis and repair, emerges as a significant player in the narrative of cancer development. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) is a major step in producing DNA. Numerous malignancies, including those of the breast, colon, lung, and ovary, have been linked to dysregulation of TS activity. Overexpression or mutations of TS lead to uncontrolled cell proliferation and tumorigenesis molecular interactions and signalling pathways involving TS come under scrutiny, revealing the nuanced connections that propel its involvement in cancer progression. Beyond overexpression and mutations, there emerges a subtle layer of regulation that involves microRNAs (miRNAs). These tiny particles attach to the TS messenger RNA, causing translational repression or its degradation, which in turn affects TS activity. Moving towards the therapeutic realm, thymidylate synthase inhibition acts as a promising anti-cancer strategy. Targeting TS with small-molecule inhibitors could provide a novel approach to treat various cancers. By reducing the number of available nucleotides, TS inhibition would slow down or halt cancer cell division, thus depriving the tumor of the building blocks required for its proliferation and growth. The aim is to assess the viability and effectiveness of targeting TS to halt or slow down cancer progression. There is growing evidence that, in comparison to traditional TS inhibitors, few novel antifolate TS inhibitors are effective against a wider variety of neoplasms, such as lung carcinomas. It has been discovered that TS inhibitors increase cancer tissues' sensitivity to chemotherapy and radiation, increasing their vulnerability to these treatments. This article aims to provide a comprehensive insight into TS, examining its cellular details, detailing the heterocyclic moieties and molecular foundations, and providing a promising future outlook.

Mitochondria-Targeted Thymidylate Synthase Inhibitor Based on Fluorescent Molecularly Imprinted Polymers for Tumor Antimetabolic Therapy.

Antimetabolites targeting thymidylate synthase (TS), such as 5-fluorouracil and capecitabine, have been widely used in tumor therapy in the past decades. Here, we present a strategy to construct mitochondria-targeted antimetabolic therapeutic nanomedicines based on fluorescent molecularly imprinted polymers (FMIP), and the nanomedicine was denoted as Mito-FMIP. Mito-FMIP, synthesized using fluorescent dye-doped silica as the carrier and amino acid sequence containing the active center of TS as the template peptide, could specifically recognize and bind to the active site of TS, thus inhibiting the catalytic activity of TS, and therefore hindering subsequent DNA biosynthesis, ultimately inhibiting tumor growth. The imprinting factor of FMIP reached 2.9, and the modification of CTPB endowed Mito-FMIP with the ability to target mitochondria. In vitro experiments demonstrated that Mito-FMIP was able to efficiently aggregate in mitochondria and inhibit CT26 cell proliferation by 59.9%. The results of flow cytometric analysis showed that the relative mean fluorescence intensity of Mito-FMIP accumulated in the mitochondria was 3.4-fold that of FMIP. In vivo experiments showed that the tumor volume of the Mito-FMIP-treated group was only one third of that of the untreated group. In addition, Mito-FMIP exibited the maximum emission wavelength at 682 nm, which allowed it to be used for fluorescence imaging of tumors. Taken together, this study provides a new strategy for the construction of nanomedicines with antimetabolic functions based on molecularly imprinted polymers.

Review of 5-FU resistance mechanisms in colorectal cancer: clinical significance of attenuated on-target effects.

The emergence of chemoresistant disease during chemotherapy with 5-Fluorouracil-based (5-FU-based) regimens is an important factor in the mortality of metastatic CRC (mCRC). The causes of 5-FU resistance are multi-factorial, and besides DNA mismatch repair deficiency (MMR-D), there are no widely accepted criteria for determining which CRC patients are not likely to be responsive to 5-FU-based therapy. Thus, there is a need to systematically understand the mechanistic basis for 5-FU treatment failure and an urgent need to develop new approaches for circumventing the major causes of 5-FU resistance. In this manuscript, we review mechanisms of 5-FU resistance with an emphasis on: (1) altered anabolic metabolism limiting the formation of the primary active metabolite Fluorodeoxyuridylate (5-Fluoro-2'-deoxyuridine-5'-O-monophosphate; FdUMP); (2) elevated expression or activity of the primary enzymatic target thymidylate synthase (TS); and (3) dysregulated programmed cell death as important causes of 5-FU resistance. Importantly, these causes of 5-FU resistance can potentially be overcome through the use of next-generation fluoropyrimidine (FP) polymers (e.g., CF10) that display reduced dependence on anabolic metabolism and more potent TS inhibitory activity.

Targeting Thymidylate Synthase and tRNA-Derived Non-Coding RNAs Improves Therapeutic Sensitivity in Colorectal Cancer.

Some colorectal cancer (CRC) patients are resistant to 5-fluorouracil (5-FU), and high expression levels of thymidylate synthase (TS) contribute to this resistance. This study investigated whether quercetin, a representative polyphenol compound, could enhance the effect of 5-FU in CRC cells. Quercetin suppressed TS levels that were increased by 5-FU in CRC cells and promoted the expression of p53. Quercetin also induced intracellular and mitochondrial reactive oxygen species (ROS) production and Ca2+ dysregulation in a 5-FU-independent pathway in CRC cells. Furthermore, quercetin decreased mitochondrial membrane potential in CRC cells and inhibited mitochondrial respiration. Moreover, quercetin regulated the expression of specific tiRNAs, including tiRNAHisGTG, and transfection of a tiRNAHisGTG mimic further enhanced the apoptotic effect of quercetin in CRC cells. An enhanced sensitivity to 5-FU was also confirmed in colitis-associated CRC mice treated with quercetin. The treatment of quercetin decreased survival rates of the CRC mouse model, with reductions in the number of tumors and in the disease activity index. Also, quercetin suppressed TS and PCNA protein expression in the distal colon tissue of CRC mice. These results suggest that quercetin has the potential to be used as an adjuvant with 5-FU for the treatment of CRC.

Computational Identification of Druggable Bioactive Compounds from Catharanthus roseus and Avicennia marina against Colorectal Cancer by Targeting Thymidylate Synthase.

Colorectal cancer (CRC) is the second most common cause of death worldwide, affecting approximately 1.9 million individuals in 2020. Therapeutics of the disease are not yet available and discovering a novel anticancer drug candidate against the disease is an urgent need. Thymidylate synthase (TS) is an important enzyme and prime precursor for DNA biosynthesis that catalyzes the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) that has emerged as a novel drug target against the disease. Elevated expression of TS in proliferating cells promotes oncogenesis as well as CRC. Therefore, this study aimed to identify potential natural anticancer agents that can inhibit the activity of the TS protein, subsequently blocking the progression of colorectal cancer. Initially, molecular docking was implied on 63 natural compounds identified from Catharanthus roseus and Avicennia marina to evaluate their binding affinity to the desired protein. Subsequently, molecular dynamics (MD) simulation, ADME (Absorption, Distribution, Metabolism, and Excretion), toxicity, and quantum chemical-based DFT (density-functional theory) approaches were applied to evaluate the efficacy of the selected compounds. Molecular docking analysis initially identified four compounds (PubChem CID: 5281349, CID: 102004710, CID: 11969465, CID: 198912) that have better binding affinity to the target protein. The ADME and toxicity properties indicated good pharmacokinetics (PK) and toxicity ability of the selected compounds. Additionally, the quantum chemical calculation of the selected molecules found low chemical reactivity indicating the bioactivity of the drug candidate. The global descriptor and HOMO-LUMO energy gap values indicated a satisfactory and remarkable profile of the selected molecules. Furthermore, MD simulations of the compounds identified better binding stability of the compounds to the desired protein. To sum up, the phytoconstituents from two plants showed better anticancer activity against TS protein that can be further developed as an anti-CRC drug.

FOXM1 inhibitor, Siomycin A, synergizes and restores 5-FU cytotoxicity in human cholangiocarcinoma cell lines via targeting thymidylate synthase

Aims5-Fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor, has been used as the first-line chemotherapeutic drug for cholangiocarcinoma (CCA). The side effects and drug resistance have developed the limits of the clinical application of 5-FU in CCA treatment. Upregulation of Forkhead box M1 (FOXM1) and TS were shown to play a significant role in 5-FU resistance. In this study, the effect of Siomycin A (SioA), a FOXM1 inhibitor, on enhancing 5-FU cytotoxicity and reversing 5-FU resistance in CCA cell lines were demonstrated. Main methodsHuman CCA cell lines, KKU-100 and KKU-213A were used. Cell viability was determined using MTT assay. Expression of FOXM1 and TS proteins were determined using Western blotting. FOXM1 mRNA expression was quantitated using real-time PCR. The combination and dose reduction (DRI) were analyzed according to the Chou and Talalay method. Key findingSingle drug treatment of 5-FU and SioA effectively inhibited CCA cell growth in dose and time dependent fashions. The two CCA cell lines had different responses to 5-FU but exhibited similar sensitivity to SioA. FOXM1 and TS expression were increased in the 5-FU treated cells but were suppressed in the SioA treated cells. A direct binding of SioA, to TS and 5,10-methylene-tetrahydrofolate as an inactive ternary complex was simulated. The combined treatment of 5-FU with SioA showed a synergistic effect with a high DRI and restored 5-FU sensitivity in the 5-FU resistant cells. SignificanceTargeting FOXM1 using SioA in combination with 5-FU might be a strategy to overcome the 5-FU resistance in CCA.

Targeting Thymidylate Synthase Enhances the Chemosensitivity of Triple-Negative Breast Cancer Towards 5-FU-Based Combinatorial Therapy.

BackgroundThe ongoing treatment modalities for breast cancer (BC) primarily rely on the expression status of ER, PR and HER-2 receptors in BC tissues. Our strategy of chemosensitization provides new insights to counter chemoresistance, a major obstacle that limits the benefits of chemotherapy of mammary cancers.MethodsBy utilizing a murine breast cancer model employing NSG mice bearing orthotopic triple-negative breast cancer (TNBC) xenografts, we have evaluated the ability of phytochemical curcumin in chemosensitizing BC to 5-Fluorouracil (5-FU) chemotherapy and the differential modulations of cellular events in response to this strategy, independent of their receptor status.ResultsA significant synergistic antitumor potential was observed in the murine model with a sub-optimal dose treatment of 5-FU plus curcumin, as evaluated by a reduction in the tumor-related parameters. We authenticated the pivotal role of thymidylate synthase (TS) in regulating the 5-FU–curcumin synergism using the TNBC pre-clinical model. Our study also confirmed the pharmacological safety of this chemotherapeutic plus phytoactive combination using acute and chronic toxicity studies in Swiss albino mice. Subsequently, the molecular docking analysis of curcumin binding to TS demonstrated the affinity of curcumin towards the cofactor-binding site of TS, rather than the substrate-binding site, where 5-FU binds. Our concomitant in vivo and in silico evidence substantiates the superior therapeutic index of this combination.ConclusionThis is the first-ever pre-clinical study portraying TS as the critical target of combinatorial therapy for mammary carcinomas and therefore we recommend its clinical validation, especially in TNBC patients, who currently have limited therapeutic options.

Synthesis of New 1, 3, 4-Oxadiazole-Incorporated 1, 2, 3-Triazole Moieties as Potential Anticancer Agents Targeting Thymidylate Synthase and Their Docking Studies.

Thymidylate synthase (TS) has emerged as a hot spot in cancer treatment, as it is directly involved in DNA synthesis. In the present article, nine hybrids containing 1,2,3-triazole and 1,3,4-oxadiazole moieties (6–14) were synthesized and evaluated for anticancer and in vitro thymidylate synthase activities. According to in silico pharmacokinetic studies, the synthesized hybrids exhibited good drug likeness properties and bioavailability. The cytotoxicity results indicated that compounds 12 and 13 exhibited remarkable inhibition on the tested Michigan Cancer Foundation (MCF-7) and Human colorectal Carcinoma (HCT-116) cell lines. Compound 12 showed four-fold inhibition to a standard drug, 5-fluoruracil, and comparable inhibition to tamoxifen, whereas compound 13 exerted five-fold activity of tamoxifen and 24-fold activity of 5-fluorouracil for MCF-7 cells. Compounds 12 and 13 inhibited thymidylate synthase enzyme, with an half maximal inhibitory concentration, IC50 of 2.52 µM and 4.38 µM, while a standard drug, pemetrexed, showed IC50 = 6.75 µM. The molecular docking data of compounds 12 and 13 were found to be in support of biological activities data. In conclusion, hybrids (12 and 13) may inhibit thymidylate synthase enzyme, which could play a significant role as a chemotherapeutic agent.

MicroRNA-375-3p enhances chemosensitivity to 5-fluorouracil by targeting thymidylate synthase in colorectal cancer.

Resistance to chemotherapy is a major challenge for the treatment of patients with colorectal cancer (CRC). Previous studies have found that microRNAs (miRNAs) play key roles in drug resistance; however, the role of miRNA‐373‐3p (miR‐375‐3p) in CRC remains unclear. The current study aimed to explore the potential function of miR‐375‐3p in 5‐fluorouracil (5‐FU) resistance. MicroRNA‐375‐3p was found to be widely downregulated in human CRC cell lines and tissues and to promote the sensitivity of CRC cells to 5‐FU by inducing colon cancer cell apoptosis and cycle arrest and by inhibiting cell growth, migration, and invasion in vitro. Thymidylate synthase (TYMS) was found to be a direct target of miR‐375‐3p, and TYMS knockdown exerted similar effects as miR‐375‐3p overexpression on the CRC cellular response to 5‐FU. Lipid‐coated calcium carbonate nanoparticles (NPs) were designed to cotransport 5‐FU and miR‐375‐3p into cells efficiently and rapidly and to release the drugs in a weakly acidic tumor microenvironment. The therapeutic effect of combined miR‐375 + 5‐FU/NPs was significantly higher than that of the individual treatments in mouse s.c. xenografts derived from HCT116 cells. Our results suggest that restoring miR‐375‐3p levels could be a future novel therapeutic strategy to enhance chemosensitivity to 5‐FU.

In vitro apoptotic effect on human lymphatic filarial parasite by piperidine derivatives and thymidine reversal study.

A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.

A novel intraperitoneal therapy for gastric cancer with DFP-10825, a unique RNAi therapeutic targeting thymidylate synthase, in a peritoneally disseminated xenograft model.

PurposeIn advanced gastric cancer, peritoneal dissemination is a life‐threatening mode of metastasis. Since the treatment options with conventional chemotherapy remain limited, any novel therapeutic strategy that could control such metastasis would improve the outcome of treatment. We recently developed a unique RNA interference therapeutic regimen (DFP‐10825) consisting of short hairpin RNA against thymidylate synthase (TS shRNA) and cationic liposomes. The treatment with DFP‐10825 has shown remarkable antitumor activity in peritoneally disseminated human ovarian cancer–bearing mice via intraperitoneal administration. In this study, we expanded DFP‐10825 to the treatment of peritoneally disseminated gastric cancer.MethodsDFP‐10825 was administered intraperitoneally into mice with intraperitoneally implanted human gastric cancer cells (MKN45 or NCI‐N87). Antitumor activity and host survival benefits were monitored. Intraperitoneal distribution of fluorescence‐labeled DFP‐10825 was monitored in this MKN45 peritoneally disseminated mouse model.ResultsIntraperitoneal injection of DFP‐10825 suppressed tumor growth in two peritoneally disseminated cancer models (MKN45 and NCI‐N87) and increased the survival time of the MKN45 model without severe side effects. Throughout the treatment regimen, no significant body weight loss was associated with the administration of DFP‐10825. Interestingly, after intraperitoneal injection, fluorescence‐labeled DFP‐10825 retained for more than 72 hours in the peritoneal cavity and selectively accumulated in disseminated tumors.ConclusionsIntraperitoneal injection of DFP‐10825 demonstrated effective antitumor activity without systemic severe adverse effects via the selective delivery of RNAi molecules into disseminated tumors in the peritoneal cavity. Our current study indicates that DFP‐10825 could become an alternative option to improve the outcomes of patients with peritoneally disseminated gastric cancer.

SPOT-007 Identifying novel combinatorial synthetic lethal vulnerabilities in KRAS-driven lung cancer

IntroductionDe-differentiation is a highly lethal feature of aggressive breast cancers (BC), and is achieved through the epithelial-to-mesenchymal transition (EMT) and the cancer stem cell (CSC) programs. Targeting the mechanisms controlling BC de-differentiation can lead to more effective therapeutics. Recent studies indicated that nucleotide metabolism can regulate cancer stemness and EMT. Here we investigated the expression of the nucleotide metabolism enzyme and drug target thymidylate synthase (TS) in the BC subtypes and analysed its impact on BC de-differentiation.Material and methodsCells with TS knockdown and overexpression were tested in vitro and in vivo. Proteins were analysed by western blot, FACS and ELISA. Differential gene expression in TS-deficient cells was determined by RNA-seq. Immunohistochemistry (IHC) was used to stain samples from patients with different BC subtypes.Results and discussionsTS mRNA expression was found to be significantly differentially expressed among the BC subtypes, exhibiting the highest levels in aggressive triple-negative BC (TNBC). shRNA-mediated TS knockdown in TNBC cell lines (n=3) increased the population of differentiated cells (CD24high) and strongly attenuated the stem-like phenotype, like the formation of mammospheres from single cells and the migration in a cell culture wound. TS-deficient cells also showed an altered ability to form metastasis in vivo, consistent with previous observations in EMT-repressed BC cells. A rescue experiment performed by overexpressing either a wild-type or catalytically inactive TS indicated that the enzymatic activity was essential for the maintenance of the BCSC phenotype. Along with a strong repression of EMT-signature genes, RNA-seq profiling indicated a reduction of inflammatory and NF-κB signalling pathways in TS deficient cells, which dramatically reduced IL-1β production and secretion. A TS-specific gene signature was generated, which significantly associated with worst survival in BC patients. IHC staining on FFPE samples from a series of BC patients (n=120) confirmed higher TS expression in tumours that were poorly differentiated and in TNBC.ConclusionWe discovered a novel role for the TS enzyme in the maintenance of a de-differentiated and stem-like state of BC. These findings may not only open the possibility to study in-depth the role of nucleotide metabolism at the crossroad between proliferation and differentiation, but may provide the rationale for novel drug combinations with TS-inhibiting agents for the treatment of BC.

SPOT-003 Thymidylate synthase maintains the undifferentiated state of aggressive breast cancers

Introduction De-differentiation is a highly lethal feature of aggressive breast cancers (BC), and is achieved through the epithelial-to-mesenchymal transition (EMT) and the cancer stem cell (CSC) programs. Targeting the mechanisms controlling BC de-differentiation can lead to more effective therapeutics. Recent studies indicated that nucleotide metabolism can regulate cancer stemness and EMT. Here we investigated the expression of the nucleotide metabolism enzyme and drug target thymidylate synthase (TS) in the BC subtypes and analysed its impact on BC de-differentiation. Material and methods Cells with TS knockdown and overexpression were tested in vitro and in vivo. Proteins were analysed by western blot, FACS and ELISA. Differential gene expression in TS-deficient cells was determined by RNA-seq. Immunohistochemistry (IHC) was used to stain samples from patients with different BC subtypes. Results and discussions TS mRNA expression was found to be significantly differentially expressed among the BC subtypes, exhibiting the highest levels in aggressive triple-negative BC (TNBC). shRNA-mediated TS knockdown in TNBC cell lines (n=3) increased the population of differentiated cells (CD24high) and strongly attenuated the stem-like phenotype, like the formation of mammospheres from single cells and the migration in a cell culture wound. TS-deficient cells also showed an altered ability to form metastasis in vivo, consistent with previous observations in EMT-repressed BC cells. A rescue experiment performed by overexpressing either a wild-type or catalytically inactive TS indicated that the enzymatic activity was essential for the maintenance of the BCSC phenotype. Along with a strong repression of EMT-signature genes, RNA-seq profiling indicated a reduction of inflammatory and NF-κB signalling pathways in TS deficient cells, which dramatically reduced IL-1β production and secretion. A TS-specific gene signature was generated, which significantly associated with worst survival in BC patients. IHC staining on FFPE samples from a series of BC patients (n=120) confirmed higher TS expression in tumours that were poorly differentiated and in TNBC. Conclusion We discovered a novel role for the TS enzyme in the maintenance of a de-differentiated and stem-like state of BC. These findings may not only open the possibility to study in-depth the role of nucleotide metabolism at the crossroad between proliferation and differentiation, but may provide the rationale for novel drug combinations with TS-inhibiting agents for the treatment of BC.

Anticancer activity of the intraperitoneal-delivered DFP-10825, the cationic liposome-conjugated RNAi molecule targeting thymidylate synthase, on peritoneal disseminated ovarian cancer xenograft model.

IntroductionPeritoneal disseminated ovarian cancer is one of the most difficult cancers to treat with conventional anti-cancer drugs and the treatment options are very limited, although an intraperitoneal (ip) paclitaxel has shown some clinical benefit. Therefore, treatment of peritoneal disseminated ovarian cancer is a highly unmet medical need and it is urgent to develop a new ip delivered drug regulating the fast DNA synthesis.MethodsWe developed a unique RNAi molecule consisting of shRNA against the thymidylate synthase (TS) and a cationic liposome (DFP-10825) and tested its antitumor activity and PK profile in peritoneally disseminated human ovarian cancer ascites models by the luciferase gene-transfected SCID mice. DFP-10825 alone, paclitaxel alone or combination with DFP-10825 and paclitaxel were administered in an ip route to the tumor-bearing mice. The TS expression level was measured by conventional RT-PCR. The anti-tumor activity and host survival benefit by DFP-10825 treatment on tumor-bearing mice were observed as resulting from the specific TS mRNA knock-down in tumors.ResultsDFP-10825 alone significantly suppressed the growth of SKOV3-luc tumore ascites cells and further extended the survival time of these tumor-bearing mice. Combination with the ip paclitaxel augmented the antitumor efficacy of DFP-10825 and significantly prolonged the survival time in the tumor-bearing mice. Short-hairpin RNA for TS (TS shRNA) levels derived from DFP-10825 in the ascetic fluid were maintained at a nM range across 24 hours but not detected in the plasma, suggesting that TS shRNA is relatively stable in the peritoneal cavity, to be able to exert its anti-tumor activity, but not in blood stream, indicating little or no systemic effect.ConclusionCollectively, the ip delivery of DFP-10825, TS shRNA conjugated with cationic liposome, shows a favorable antitumor activity without systemic adverse events via the stable localization of TS shRNA for a sufficient time and concentration in the peritoneal cavity of the peritoneally disseminated human ovarian cancer-bearing mice.

MicroRNA-330 inhibited cell proliferation and enhanced chemosensitivity to 5-fluorouracil in colorectal cancer by directly targeting thymidylate synthase.

Colorectal cancer (CRC) is the third most common cancer in males and the second most common in females, worldwide. Currently, 5-fluorouracil (5-FU)-mediated chemotherapy is the adjuvant treatment for patients with CRC following surgical resection. However, a number of patients with CRC develop 5-FU resistance, which is a major challenge for the effective treatment of cancer. Therefore, it is important to investigate the molecular mechanisms underlying chemoresistance and the therapeutic treatments that may improve the treatment of CRC. The present study demonstrated that microRNA (miR)-330 was significantly downregulated in CRC tissues and cell lines. Ectopic miR-330 expression decreased cell proliferation and enhanced cell chemosensitivity to 5-FU via the cell apoptosis pathway in CRC. In addition, thymidylate synthase (TYMS) was revealed to be a direct target gene of miR-330 in CRC. Knockdown of TYMS inhibited CRC cell proliferation, and enhanced cell chemosensitivity to 5-FU by promoting cell apoptosis. In conclusion, the results of the present study indicated that miR-330 targeted TYMS to inhibit the proliferation and enhance the chemosensitivity of CRC cells to 5-FU by promoting cell apoptosis. The present study provided important insight into the molecular mechanism underlying 5-FU-mediated chemoresistance and a novel therapeutic strategy for the enhancement of efficacy in CRC treatment.

Abstract B16: Activation of NRF2 and adaptive resistance to chemotherapy

Abstract Nuclear factor-erythroid-2-related factor 2 (NRF2), a member of the cap ‘n’ collar family of bZIP transcription factors, confers protection against oxidative and electrophilic stress. NRF2 is of great interest in cancer research, due to its role in response to chemotherapy, including the class of drugs targeting thymidylate synthase (TYMS). It has long been known that inhibition of TYMS leads to depletion of thymidine levels and the onset of programmed cell death, deriving from the enzyme's function as the sole de novo source of thymidine for DNA replication and repair. Exposing cells to TYMS inhibitors such as fluoropyrimidine antimetabolites (5-fluorouracil, or FUra; 5'-fluoro-2'-deoxyuridine, or FdUrd), as well as anti-folate analogs (raltitrexed, or RTX), induce intracellular concentrations of reactive oxygen species, which are a primary cause of drug-mediated toxicity. This prompted our focus on assessing the impact of NRF2 on cellular response to TYMS inhibitors. Using human colon tumor-derived cell line HCT116, we have shown by gene expression profiling that drug exposure induces expression of a number of genes that are modulated by NRF2. Quantitative PCR assays of several colon tumor cell lines verified that FUra, FdUrd, and RTX induce transcription of several genes known to be NRF2-targets, including AKR1B10, ALDH3A1, HSPB8, HMOX1, and SERPINE1, among others. Such induction mirrors that in response to the known NRF2 activator tert-butylhydroquinone (tBHQ). NRF2 protein concentration and nuclear localization are increased by FdUrd treatment, though not to the same extent as with tBHQ. Reporter gene constructs were used to show that both FdUrd and tBHQ induce transcription mediated by the NRF2-binding antioxidant response element (ARE). Furthermore, chromatin-immunoprecipitation experiments revealed that TYMS inhibitors promote occupancy of the ARE regions of several genes by NRF2; again, tBHQ had a much greater effect. Further analysis showed an association between gene induction by TYMS inhibitors and patterns of histone acetylation within the NRF2-binding ARE regions. Finally, we observed that increases in the apoptotic index following exposure to TYMS inhibitors were greater in cells in which the transcription factor was subjected to siRNA-mediated “knockdown” or CRISPR/Cas9-mediated “knockout”, indicating that reduced NRF2 expression sensitizes cells to TYMS inhibitors. Overall, we conclude that TYMS inhibitors activate NRF2 and its downstream target genes, thereby constraining drug response. Reducing such activation of NRF2 or its consequences may be an effective strategy to sensitizing tumor cells to chemotherapy. Citation Format: Sarah A. Clinton, Karen W. Barbour, Franklin G. Berger. Activation of NRF2 and adaptive resistance to chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B16.

C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins

Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5′-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work.

Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance.

Our current understanding of the mechanisms of action of antitumor agents and the precise mechanisms underlying drug resistance is that these two processes are directly linked. Moreover, it is often possible to delineate chemoresistance mechanisms based on the specific mechanism of action of a given anticancer drug. A more holistic approach to the chemoresistance problem suggests that entire metabolic pathways, rather than single enzyme targets may better explain and educate us about the complexity of the cellular responses upon cytotoxic drug administration. Drugs, which target thymidylate synthase and folate-dependent enzymes, represent an important therapeutic arm in the treatment of various human malignancies. However, prolonged patient treatment often provokes drug resistance phenomena that render the chemotherapeutic treatment highly ineffective. Hence, strategies to overcome drug resistance are primarily designed to achieve either enhanced intracellular drug accumulation, to avoid the upregulation of folate-dependent enzymes, and to circumvent the impairment of DNA repair enzymes which are also responsible for cross-resistance to various anticancer drugs. The current clinical practice based on drug combination therapeutic regimens represents the most effective approach to counteract drug resistance. In the current paper, we review the molecular aspects of the activity of TS-targeting drugs and describe how such mechanisms are related to the emergence of clinical drug resistance. We also discuss the current possibilities to overcome drug resistance by using a molecular mechanistic approach based on medicinal chemistry methods focusing on rational structural modifications of novel antitumor agents. This paper also focuses on the importance of the modulation of metabolic pathways upon drug administration, their analysis and the assessment of their putative roles in the networks involved using a meta-analysis approach. The present review describes the main pathways that are modulated by TS-targeting anticancer drugs starting from the description of the normal functioning of the folate metabolic pathway, through the protein modulation occurring upon drug delivery to cultured tumor cells as well as cancer patients, finally describing how the pathways are modulated by drug resistance development. The data collected are then analyzed using network/netwire connecting methods in order to provide a wider view of the pathways involved and of the importance of such information in identifying additional proteins that could serve as novel druggable targets for efficacious cancer therapy.

Diaza-anthracene Antibiotics from a Freshwater-Derived Actinomycete with Selective Antibacterial Activity toward Mycobacterium tuberculosis

Multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis are resistant to first- and second-line drug regimens and resulted in 210,000 fatalities in 2013. In the current study, we screened a library of aquatic bacterial natural product fractions for their ability to inhibit this pathogen. A fraction from a Lake Michigan bacterium exhibited significant inhibitory activity, from which we characterized novel diazaquinomycins H and J. This antibiotic class displayed an in vitro activity profile similar or superior to clinically used anti-tuberculosis agents and maintained this potency against a panel of drug-resistant M. tuberculosis strains. Importantly, these are among the only freshwater-derived actinomycete bacterial metabolites described to date. Further in vitro profiling against a broad panel of bacteria indicated that this antibiotic class selectively targets M. tuberculosis. Additionally, in the case of this pathogen we present evidence counter to previous reports that claim the diazaquinomycins target thymidylate synthase in Gram-positive bacteria. Thus, we establish freshwater environments as potential sources for novel antibiotic leads and present the diazaquinomycins as potent and selective inhibitors of M. tuberculosis.