HL-60 Tumor Research Articles

In vitro assessment of the cytotoxicity of Gallium(III) complexes with Isoniazid-Derived Hydrazones: Effects on clonogenic survival of HCT-116 cells

Cancer cells have high iron demand to mediate their rapid proliferation. Aiming to obtain cytotoxic compounds with potential iron metabolism disturbance in malignant cells, [Ga(HAPIH)(APIH)](NO3)2⋅2H2O (1) and [Ga(HPAmIH)(PAmIH)](NO3)2⋅2H2O (2) were synthesized with the iron chelators 2-acetylpyridine- and 2-pyridineformamide isonicotinoyl hydrazone (HAPIH and HPAmIH, respectively) and gallium(III), an iron(III) mimetic. The hydrazones and their gallium(III) complexes were assayed for their action against HL-60 (leukemia), MCF-7 (breast cancer), HCT-116 (colorectal carcinoma) and PC3 (prostate cancer) tumor cell lines, as well as non-malignant Human Embryonic Kidney293 (HEK-293) cells. HAPIH and its complex 1 were the most cytotoxic compounds, with HCT-116 being the most susceptible line (IC50 = 1.6 μM for HAPIH and 0.4 μM for complex 1). Moreover, the compounds proved to be at least 25-fold less toxic to HEK-293 than to the tumor cells. According to clonogenic survival assays, the relative number of colonies formed by HCT-116 cells was reduced by around 95% after pretreatment with HAPIH and 1 at their IC90 concentrations (18 μM and 4 μM, respectively). Both compounds were also able to inhibit the cell cycle and increased the subdiploid DNA content of HCT-116 after 24 h treatment, which suggests they possess a pro-apoptotic potential.

Toxicological Studies of Czech Beers and Their Constituents

Background: Czech beers are unique because they are brewed using specific technology at a particular latitude and for being entirely produced in the area of the Czech Republic. The purpose of this work is the evaluation of toxicological effects of a variety of freeze-dried Czech beers, their raw materials (malts, hops and yeast) and processed-beer (wort, hopped wort and young beer). Methods: In vivo assays to evaluate the safety and protective effects in the Drosophila melanogaster eukaryotic system, and the in vitro evaluations of chemopreventive and DNA damage activity using the HL-60 tumour human cell line were carried out. Results: The safe effects for all the analysed substances and general protective effects against H2O2 were shown both at the individual and genomic level in the Drosophila animal model, with some exceptions. Moreover, all the substances were able to inhibit the tumour cell growth and to induce DNA damage in the HL-60 cells at different levels (proapoptotic, single/double strands breaks and methylation status). Conclusions: The promising effects shown by freeze-dried Czech beers due to their safety, protection against a toxin, chemopreventive potential and the induction of DNA damage in tumour cells, allow the proposition of Czech beer as a beverage with nutraceutic potential.

The Synthesis of Bicyclo[4.2.1]nona-2,4,7-trienes by [6π + 2π]-Cycloaddition of 1-Substituted 1,3,5-Cycloheptatrienes Catalyzed by Titanium and Cobalt Complexes.

The [6π + 2π]-cycloaddition of alkynes to 1-methyl-, propyl-, benzyl-, and hydroxymethyl-substituted 1,3,5-cycloheptatrienes in the presence of catalytic systems Ti(acac)2Cl2-Et2AlCl and Co(acac)2(dppe)/Zn/ZnI2 was performed for the first time to give practically valuable bicyclo[4.2.1]nona-2,4,7-trienes in high yields (72-88%). The structures of the obtained bicyclic compounds were reliably proved by NMR methods and X-ray diffraction analysis. The newly synthesized bicycles have been investigated for their in vitro cytotoxic activity against Jurkat, K562, U937, and HL60 tumor and normal cell lines and induction of apoptosis.

Biological Effects of Food Coloring in In Vivo and In Vitro Model Systems.

(1) Background: The suitability of certain food colorings is nowadays in discussion because of the effects of these compounds on human health. For this reason, in the present work, the biological effects of six worldwide used food colorings (Riboflavin, Tartrazine, Carminic Acid, Erythrosine, Indigotine, and Brilliant Blue FCF) were analyzed using two model systems. (2) Methods: In vivo toxicity, antitoxicity, and longevity assays using the model organism Drosophila melanogaster and in vitro cytotoxicity, DNA fragmentation, and methylation status assays using HL-60 tumor human cell line were carried out. (3) Results: Our in vivo results showed safe effects in Drosophila for all the food coloring treatments, non-significant protective potential against an oxidative toxin, and different effects on the lifespan of flies. The in vitro results in HL-60 cells, showed that the tested food colorings increased tumor cell growth but did not induce any DNA damage or modifications in the DNA methylation status at their acceptable daily intake (ADI) concentrations. (4) Conclusions: From the in vivo and in vitro studies, these results would support the idea that a high chronic intake of food colorings throughout the entire life is not advisable.

Synthesis and Anticancer Activity of Novel 9-O-Substituted Berberine Derivatives.

Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values ranging from 0.7 to 16.7 µM for HL-60 cells and 36 to >200 µM for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G2/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC50 value of 0.7 µM and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative.

Structurally diverse diterpenoids from Isodon ternifolius collected from three regions

Phytochemical investigation of the aerial parts of Isodon ternifolius from three regions in Yunnan Province afforded 9 new diterpenoids, ternifoliusins A–I (1–9), together with 26 known diterpenoids. Among them, ternifoliusins A–D possess rare 6,7:8,15-diseco-7,20-olide-6,8-cyclo-ent-kaurane diterpenoid scaffold. The structures of all compounds were determined by comprehensive spectroscopic analysis, as well as quantum chemical calculation methods. Moreover, the molecular constitutions and configurations of ternifoliusins A, E, and F (1, 5, 6) were confirmed by single-crystal X-ray diffraction analysis. Ten compounds were assayed for their cytotoxic activity against the HL-60, SMMC-7721, A-549, MCF-7, and SW-480 human tumor cell lines, and compounds 13, 17, 19, 20, 31 and 34 were demonstrated to be active against all of the five human tumor cell lines.

Cytotoxic butenolides and diphenyl ethers from the endophytic fungus Pestalotiopsis sp.

Chemical investigation of the plant endophytic fungus Pestalotiopsis sp. afforded two new butenolides, pestalolides B and C (1 and 2), two new diphenyl ethers, pestalotethers E and F (3 and 4), together with seven known compounds (5–11). Their structures were established by extensive spectroscopic analyses and the absolute configuration of 1 was further confirmed by ECD calculation. Structurally, compound 1 represents the first example of a new type of butenolide derivatives, bearing an unprecedented carbon scaffold having 13 carbon atoms. Compounds 1–4 were evaluated for the cytotoxic activities, and compound 1 showed remarkable inhibitory potency against human tumor HL60, U87MG, MDA-MB-231, and HEP-3B cell lines, with IC50 values of 1.42, 5.90, 5.90, and 4.29 μM, respectively.

Knockout Tumor Microenvironment HO-1 Neutralizes Myeloid-Derived Suppressor Cells and Enhances the Antitumor Effect of PD-1 Inhibition in Murine Models of Acute Myeloid Leukemia

Background/Aims: Myeloid-derived suppressor cells (MDSCs) has been shown to be involved in tumor immune escape mechanisms and B cell-specific immunity in Acute myeloid leukemia (AML) patients. In our previous study shown that HO-1 protein have immunomodulatory activity including targeting of immune suppressor cells in the tumor microenvironment. Thus, we decided to assess whether HO-1 could enhance anti-PD-1 treatment and investigate those alterations in the immunosuppressive tumor microenvironment that contribute to the combined antitumor activity.Method: We utilized C57/BL6 (HO-1 knockout) mouse were radiation with 6.5GY once in a row to repress residual immunity. Malignant tumor HL-60 cells respectively (1×107 cells) per animal were injected subcutaneously into the right abdomen. The xenograft mouse models of AML were euthanised on the 14th day after treatment with a PD-1 inhibitor once a day (20 mg/kg). The tumor volumes were measured and calculated by ruler. Survival curve of individual groups was evaluated from the first day of treatment until death using Kaplan-Meier curves.Results: HO-1 gene knockout enhanced the antitumor effect of PD-1 inhibition in xenograft mouse models of AML by reducing tumor growth and increasing survival. HO-1 gene knockout inhibited the immunosuppressive function of both polymorphonuclear (PMN)- and monocytic-myeloid derived suppressor cell (M-MDSC) populations. Analysis of MDSC response to HO-1 gene knockout revealed significantly reduced arginase-1, iNOS, and COX-2 levels, suggesting potential mechanisms for the altered function. We also observed significant alterations in cytokine/chemokine release in vivo with a shift toward a tumor-suppressive microenvironment.Conclusions: Our results demonstrate that HO-1 gene knockout enhances the antitumor effect of PD-1 targeting through functional inhibition of MDSCs and a transition away from an immune-suppressive tumor microenvironment.[Key words] HO-1 gene knockout; PD-1 inhibitor; tumor microenvironment; MDSCs; AML DisclosuresNo relevant conflicts of interest to declare.

Proliferation effects of cinnamon extract on human HeLa and HL-60 tumor cell lines.

To investigate the possible anti-cancer properties of cinnamon extract on two human tumor cell lines, HeLa cells and HL-60 cells. Two human tumor cell lines, HeLa cells and HL-60 cells, were exposed to increased concentrations of an extract prepared from cinnamon. The cell proliferation and cell cycle distribution were evaluated using MTT assay and flow cytometry, respectively. The possible action mechanism was also investigated by Western blot. The results showed that cinnamon extract strongly inhibited tumor cell proliferation in a dose-dependent manner and exhibited dramatic increases in the percentage of cells in G2/M in parallel with exposure to increasing concentration of cinnamon extract. The Western blot results showed that cinnamon extract reduced the cyclin A, cyclin B1, ERK2, and p-ERK proteins expression. Our study suggested that cinnamon extract inhibit the tumor cell survival by both down-regulated their target cell cycle regulation molecules and mitosis regulation molecules.

Abstract 2561: Fully human immunoglobulin heavy chain only-derived CD33 CAR for the treatment of acute myeloid leukemia

Abstract CD33 antigen is a promising target expressed on non-solid cancers, including acute myeloid leukemia (AML). Present investigative approaches to treatment of CD33-positive AML include antibody drug conjugates (My96, Mylotarg®) and CART cells incorporating CD33-targeting domains derived from a humanized scFv. Here, we designed a chimeric antigen receptor utilizing targeting domain derived from a fully human CD33 fragment variable heavy chain sequence, termed CAR33VH, and examined its in vitro and in vivo potency against AML. Primary human CD4+ CD8+ T cells derived from three healthy donors were transduced with lentiviral constructs (LV) encoding CAR33VH, or control CAR construct based on scFv My96 (My96CAR). Flow cytometric analysis revealed expression of CAR33VH at 32%-45%, and expression of My96CAR at 77% - 86%. When challenged with CD33+ AML tumor lines HL60 and MOLM-14 in vitro, both constructs demonstrated efficient target killing. CD33- tumor lines K562 and Reh were not sensitive to CAR killing, underscoring CAR specificity to CD33 antigen. Pro-inflammatory cytokines IFN gamma, TNF alpha and IL-2 in culture supernatants of CART cells incubated with CD33+ HL-60 and MOLM-14 tumors, but not with CD33- K562 cells overnight, were induced, as measured by ELISA. Long-term co-incubation assay of CART cells with HL-60 leukemia at E:T ratios 1:5 to 1: 0.04, suggested similar killing potency and persistence of CAR33VH to the positive control scFv-based CAR. In in vivo AML model, NSG mice engrafted with MOLM-14 cells stably expressing firefly luciferase, both CAR33VH, and My96CAR control were equally efficient in tumor elimination. In conclusion, CAR33VH, comprised of a fully human heavy-chain variable fragment only antigen binding domain, was efficient in tumor killing in vitro and in vivo, and may be used clinically for treatment of CD33+ hematologic malignancies. To our knowledge, this is one of the first instances demonstrating the feasibility of employing heavy chain only binder sequence in CART design. Citation Format: Dina Schneider, Ying Xiong, Weizao Chen, Zhongyu Zhu, Darong Wu, Jennifer Hwang, Dimiter S. Dimitrov, Boro Dropulic, Rimas J. Orentas. Fully human immunoglobulin heavy chain only-derived CD33 CAR for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2561.

Oxidative skeletal rearrangement of bicyclo[4.2.2]deca-2,4,7,9-tetraenes to bicyclo[4.3.1]deca-2,4,8-triene-7,10-diols and study of the antitumor activity of the products in vitro

Bicyclo[4.3.1]deca-2,4,8-triene-7,10-diols were synthesized in 76–85% yields by oxidative skeletal isomerization of the substituted bicyclo[4.2.2]deca-2,4,7,9-tetraenes of various structures on treatment with m-chloroperbenzoic acid. The structures of the obtained bicyclic unsaturated diols were reliably proven by modern spectral methods and X-ray diffraction. A high antitumor activity in vitro was found for bicyclo[4.3.1]deca-2,4,8-triene-7,10-diols against the Jurkat, K562, U937 and HL-60 tumor cell lines.

Synthesis, Pharmacological Evaluation and Docking Study of a New Modulator of Microtubule Polymerization

Objectives: The main goal of this paper was to conduct the synthesis to determine cytotoxic activity and to carry out docking studies on new LASSBio-1586 isosteres. LASSBio-1586 is a new combretastatin A4 (CA4) analogue previously identified as a simple antitumor drug candidate, able to inhibit microtubule polymerization with broad in vitro and in vivo cytotoxic activity. Methods: The new isosteres (7b-7h, 8a and 9a) were evaluated against HL-60, OVCAR-8, HCT8 and LUCENA tumor cell lines, using cytotoxic test of MTT. The tubulin polymerization assay was performed using a tubulin polymerization assay kit from cytoskeleton® and by CEREP employing a single concentration of 10 µM. Binding mode at β-tubulin colchicine binding site was stablished by blind molecular docking studies. Results: LASSBio-1920 (7h) was identified as the most potent cytotoxic compound with IC50 values ranging from 0.75 nM to 11.5 nM, although it was inactive against MDR tumor cell line LUCENA (IC50 = 80 µM). This compound presented remarkable cytotoxic selective index in comparison with CA4 and LASSBio-1586. Its ability to modulate microtubule polymerization was confirmed and its mode of interaction with colchicine binding site in β-tubulin was demonstrated. Conclusion: Compound 7h is a new isostere of LASSBio-1586 that displayed potent cytotoxic activity and better cytotoxic selectivity index and has been shown to interact with DAMA-colchicine in its co-crystal with β-tubulin. Keywords: Tubulin, cytotoxic activity, cancer, microtubule, CA4, docking study.

Two new cytotoxic steroids from the Chinese soft coral Sinularia sp.

Two new steroids, ximaosteroid E (1) and ximaosteroid F (2), along with two known related compounds (3 and 4), were isolated from the Chinese soft coral Sinularia sp. Notably, 1 possesses an uncommon dihydrofuran group. Their structures were established from extensive spectroscopic analyses and comparisons of their spectral data with those reported in the literature. The absolute configuration of 2 was determined by applying the modified Mosher’s method. In bioassay, compounds 1, 2, and 4 showed significant cytotoxicity against the HL-60 tumor cell line with IC50 values of 1.79, 4.03 and 0.69 μM, respectively.

A New Breviane Spiroditerpenoid from the Marine-Derived Fungus Penicillium sp. TJ403-1.

Marine-derived fungi are a promising and untapped reservoir for discovering structurally interesting and pharmacologically active natural products. In our efforts to identify novel bioactive compounds from marine-derived fungi, four breviane spiroditerpenoids, including a new compound, brevione O (1), and three known compounds breviones I (2), J (3), and H (4), together with a known diketopiperazine alkaloid brevicompanine G (5), were isolated and identified from an ethyl acetate extract of the fermented rice substrate of the coral-derived fungus Penicillium sp. TJ403-1. The absolute structure of 1 was elucidated by HRESIMS, one- and two-dimensional NMR spectroscopic data, and a comparison of its electronic circular dichroism (ECD) spectrum with the literature. Moreover, we confirmed the absolute configuration of 5 by single-crystal X-ray crystallography. All the isolated compounds were evaluated for isocitrate dehydrogenase 1 (IDH1) inhibitory activity and cytotoxicity, and compound 2 showed significant inhibitory activities against HL-60, A-549, and HEP3B tumor cell lines with IC50 values of 4.92 ± 0.65, 8.60 ± 1.36, and 5.50 ± 0.67 µM, respectively.

Melanogenesis-Inhibitory and Cytotoxic Activities of Chemical Constituents from the Leaves of Sauropus androgynus L. Merr. (Euphorbiaceae).

A new steroid, 20-hydroxyisofucosterol (stigmasta-5,24(28)-diene-3β,20β-diol) (7), along with six known compounds 1 - 6 were isolated from the MeOH extract of the leaves of Sauropus androgynus L. Merr. (Euphorbiaceae). The structure of new steroid was determined by HR-APCI-MS and various NMR techniques in combination with literature data. Subsequently, their anti-inflammatory, cytotoxic activities against five human cell lines, as well as inhibitory activities against the α-MSH induced melanogenesis on the B16 cell line were evaluated. As the results, steroid compounds, 6 and 7 exhibited moderate cytotoxic to HL60, AZ521, SKBR3, and A549 tumor cell lines (IC50 26.9 - 45.1 μm) with high tumor selectivity for A549 relative to WI38 cell lines (SI 2.6 and 3.0, resp.). And, flavonoid compounds, 4 and 5 exhibited superior inhibitory activities against melanogenesis (67.0 - 94.7% melanin content), even with no or low toxicity to the cells (90.1 - 99.6% cell viability) at the concentrations from 10 to 100 μm. Furthermore, Western blot analysis suggested that compound 5 could inhibit melanogenesis by suppressing the protein expressions of MITF, TRP-1, TRP-2, and tyrosinase.

Design, synthesis and cytotoxic activity of N-Modified oleanolic saponins bearing A glucosamine

A series of N-acyl, N-alkoxycarbonyl, and N-alkylcarbamoyl derivatives of 2′-deoxy-glucosyl bearing oleanolic saponins were synthesized and evaluated against HL-60, PC-3, and HT29 tumor cancer cells. The SAR studies revealed that the activity increased in order of conjugation of 2′ -amino group with carbamate > amide > urea derivatives. Lengthening the alkyl chain increased the cytotoxicity, the peak activity was found to around heptyl to nonyl substitutions. 2′-N-heptoxycarbonyl derivative 56 was found to be the most cytotoxic (IC50 = 0.76 μM) against HL-60 cells. Due to the interesting SARs of alkyl substitutions, we hypothesized that their location in the cell was different, and pursued a location study using 2′-(4″-pentynoylamino) 2′-deoxy-glucosyl OA, which suggested that these compounds distributed mainly in the cytosol.

A novel approach to oxoisoaporphine alkaloids via regioselective metalation of alkoxy isoquinolines.

Oxoisoaporphine alkaloids are conveniently prepared via direct ring metalation of alkoxy-substituted isoquinolines at C-1, followed by reaction with iodine. Subsequent Suzuki cross-coupling of the resulting 1-iodoisoquinolines to methyl 2-(isoquinolin-1-yl)benzoates and intramolecular acylation of the corresponding carboxylic acids with Eaton’s reagent afforded five alkaloids of the oxoisoaporphine type. The yield of the cyclization step strongly depends on the electrophilic properties of ring B. An alternative cyclization protocol via directed remote metalation of ester and amide intermediates was investigated thoroughly, but found to be not feasible. Two of the alkaloids showed strong cytotoxicity against the HL-60 tumor cell line.

Four Hybrid Flavan-Chalcones, Caesalpinnone A Possessing a 10,11-Dioxatricyclic [5.3.3.01,6]Tridecane-Bridged System and Caesalpinflavans A-C from Caesalpinia enneaphylla.

Caesalpinnone A (1), an unprecedented hybrid of flavan and chalcone, possessing a 10,11-dioxatricyclic [5.3.3.01,6]tridecane-bridged system, and caesalpinflavans A-C (2-4), three new hybrid flavan-chalcones, were isolated from the twigs and leaves of Caesalpinia enneaphylla. Their structures were elucidated by a combination of spectroscopic analyses and single-crystal X-ray diffraction. Caesalpinnone A showed the highest cytotoxicity against the HL-60, SMMC-7721, A-549, MCF-7, and SW-480 human tumor cell lines with an IC50 in the range of 0.54-0.87 μM.

Role of Zucchini and Its Distinctive Components in the Modulation of Degenerative Processes: Genotoxicity, Anti-Genotoxicity, Cytotoxicity and Apoptotic Effects.

Zucchini (Cucurbita pepo subsp. pepo) is a seasonal vegetable with high nutritional and medical values. Many useful properties of this fruit are attributed to bioactive compounds. Zucchini fruits (“Yellow” and “Light Green” varieties) and four distinctive components (lutein, β-carotene, zeaxanthin and dehydroascorbic acid) were selected. Firstly, the lutein, β-carotene, zeaxanthin and dehydroascorbic acid contents were determined in these fruits. Then, in order to evaluate the safety and suitability of their use, different assays were carried out: (i) genotoxicity and anti-genotoxicity tests to determine the safety and DNA-protection against hydrogen peroxide; (ii) cytotoxicity; and (iii) DNA fragmentation and Annexin V/PI (Propidium Iodide) assays to evaluate the pro-apoptotic effect. Results showed that: (i) all the substances were non-genotoxic; (ii) all the substances were anti-genotoxic except the highest concentration of lutein; (iii) “Yellow” zucchini epicarp and mesocarp exhibited the highest cytotoxic activity (IC50 > 0.1 mg/mL and 0.2 mg/mL, respectively); and (iv) “Light Green” zucchini skin induced internucleosomal DNA fragmentation, β-carotene being the possible molecule responsible for its pro-apoptotic activity. To sum up, zucchini fruit could play a positive role in human health and nutrition due to this fruit and its components were safe, able to inhibit significantly the H2O2-induced damage and exhibit anti-proliferative and pro-apoptotic activities toward HL60 (human promyelocytic leukemia cells) tumor cells. The information generated from this research should be considered when selecting potential accessions for breeding program purposes.

Design, synthesis and biological evaluation of novel 6-substituted pyrrolo [3,2-d] pyrimidine analogues as antifolate antitumor agents

A series of novel 6-substituted pyrrolo[3,2-d]pyrimidine analogues (10a, 11a-13a, 15a, 17a, 18a, 27a and 28a) have been designed and synthesized as antifolate antitumor agents. The anti-proliferative activities of these compounds against HL60, A549, H1299, Hela, HCT116 and HT29 tumor cells were evaluated. Most of the compounds exhibited micromolar anti-proliferative potencies. Compound 15a, the most potent one, has GI50 value of 0.73, 1.72, and 8.92 μM against A549, H1299 and HL60 cells, respectively. The cell cycle distribution assay displayed that 15a could increase the accumulation of G2/M-phase cells. 15a showed low potency in induction of apoptosis. However, the inhibition of A549 cell colony formation was observed. These indicated that the tumor cell death relied on the irreversible effect of 15a on clonogenicity and cell proliferation. The identification of targeted pathway of 15a implied that the anti-proliferative potencies of 15a probably act through dual inhibition of thymidylate synthase (TS) and dihydrofolate reductase (DHFR).