Transcription factor 4 (TCF4)/β-catenin complex; TCF4/β-catenin; KDM4A

When applied to Hep40, HepG2, and Huh7 cell lines, xantothricin demonstrates dose-dependent cytotoxicity with IC50 values of 0.66 μM, 0.36 μM, and 0.98 μM, respectively [1].

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Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) PKF115-584 and CGP049090 are cytotoxic against hepatoma cells but not normal hepatocytes. Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) , PKF115-584 and CGP049090 dose-dependently inhibit Tcf4/β-catenin interaction and transcriptional activity. Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) , PKF115-584 and CGP049090 caused apoptosis and cell cycle arrest in HepG2 and Huh7 cells.[1]

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Researchers describe the in silico, in vitro, and cell-based characterization of the compound Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) , an antagonist of transcription factor 4 (TCF4)/β-catenin signaling, as inhibitor of KDM4A. PKF118-310 potential inhibitor activity was discovered via virtual screening on the crystal structure of KDM4A. A peptide-based histone trimethylation assay developed in-house confirmed its potent KDM4A inhibitor activity. Its protein target was identified by cellular thermal shift assay experiments. PKF118-310 anticancer activity was observed in both liquid and solid tumor cells, and shown to have a dose- and time-dependent effect. We demonstrate the previously unreported inhibitory action of PKF118-310 on KDM4A. Our findings open up the possibility of developing the first KDM4A-specific inhibitors and derivatives[2].

Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) , PKF115-584 and CGP049090 inhibit growth of HCC xenografts in nude mice[1]
We next studied the in vivo effects of the 3 chemicals on tumor growth. HepG2 cells were injected subcutaneously into nude mice to induce tumor formation. Preliminary limited toxicity studies in nude mice suggested that all 3 chemicals were toxic at doses greater than 2 mg/kg (treated nude mice died within 1 week), whereas no observable toxicity was observed at 1 mg/kg for more than 3 weeks. We therefore used 1 mg/kg for treatment of xenografted mice. Following 35 days of tumor growth, when established xenografts were palpable, mice were intratumorally injected with each of 3 chemicals (n = 5) and control PBS (n = 5) twice weekly. Compared with vehicle control, all 3 chemicals effectively inhibited tumor growth in vivo at the beginning of the 3rd week of treatment (ANOVA, p < 0.05 for all 3 chemicals) (Fig. 5a). The tumor tissues were harvested and analyzed via TUNEL staining, and apoptotic cells were detected in tumors treated with all 3 chemicals (Fig. 5b). Additionally, tumors treated with all 3 chemicals showed reduced c-Myc, cyclin D1 and survivin expressions when immunostained with specific antibodies (representative images for PKF118-310 only are shown in Fig. 5c). These results are consistent with our in vitro observations that these chemicals inhibit Tcf4/β-catenin mediated transcriptional activity.

Cellular thermal shift assay (CETSA)[2]
HCT-116 cells were harvested and washed with PBS after treatment with Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) (10 μM) and an equal amount of DMSO, as control, for 1 h. The respective samples were suspended in PBS (1.5 mL), divided into aliquots (100 μl), and heated at different temperatures for 3 min by Thermo Mixer, followed by cooling for 3 min at 4°C. After incubation, lysis buffer (100 μl) was added to the samples and incubated for 15 min. The samples were then centrifuged at 13,000 rpm for 30 min at 4°C, the supernatant was removed, and the protein content was determined using a Bradford assay. Of the total protein extract, 20 μg was loaded on 10% SDS-PAGE. Nitrocellulose filters were stained with Ponceau red as an additional control for equal loading.

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Luciferase reporter gene assay[1]
Luciferase reporter gene assays were used to measure the ability of Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) , PKF115-584 and CGP049090 to disrupt Tcf4/β-catenin binding ability and Tcf4/β-catenin regulated transcriptional activity. The Tcf4/β-catenin binding assay was measured with the mammalian 2-hybrid system as described,26 using the pBIND/β-catenin, pACT/Tcf4 and pG5/luc plasmids that were used. Tcf4/β-catenin transcriptional reporter gene assays were performed using TCF/Luc reporter constructs, wild type pGL3-OT and mutant pGL3-OF, which were generously provided by B Vogelstein.[1]
Twenty-four hours prior to transfection, HepG2 or Huh7 cells were seeded at 3 × 104 cells/well into 24-well plates. Cells were transfected with pBIND/β-catenin (0.35 μg), pACT/Tcf4 (0.35 μg) and pG5/luc (0.1 μg) for Tcf4/β-catenin binding assay, or with wild type pGL3-OT or mutant pGL3-OF (0.7 μg) for Tcf4 transcriptional assay using Lipofectamine 2000 according to the manufacturer's instructions. The β-galactosidase expression vector (0.1 μg) was added to each transfection system to normalize the transfection efficiency. After 4 hr, medium containing transfection reagent was replaced with new culture medium containing various concentrations (0.2–1.6 μM) of each chemical. After 24 hr, cells were lysed in 100 μl of lysis buffer and 20 μl aliquots were assayed for luciferase activity using the Promega Luciferase assay system or for β-gal activity using the Promega β-gal assay system. Relative light units (RLU) were measured and normalized for transfection efficiency using β-galactosidase activity. Final RLU representing Tcf4 transcriptional activity were calculated by subtracting normalized levels obtained with pGL3-OF from those obtained with pGL3-OT.

Cell proliferation assay[2]
Cancer cell proliferation was tested with the xCELLigence system. HCT-116 cells were dispensed in 96-well plates (E-Plate, Roche) in duplicate at 2 × 104 cells/mL to evaluate cellular impedance. Impedance is dependent on confluence level. An arbitrary unit parameter, ‘Cell Index' (CI), was assigned to express confluence. Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) (10 μM and 1 μM) and DMSO were added 6 h post-seeding, in duplicate. Dynamic CI values were monitored at 30-min intervals from the time of plating until the end of the experiment. CI values were calculated and plotted on a line graph and histogram.

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Apoptosis analysis[1]
Terminal dUTP-mediated nick-end labeling (TUNEL) assays (Promega) were performed according to the manufacturer's protocol. Briefly, HepG2 or Huh7 cells were seeded in 8-chamber BD tissue culture slides at 10% confluency. Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) , PKF115-584 and CGP049090 were added to the culture medium at final concentrations of 0.15, 0.3 and 0.35 μM, respectively. After 72 hr incubation, cells were washed twice with PBS and then fixed in 4% paraformaldehyde for 25 min. Fixed cells were washed twice in PBS with 0.1% Triton X-100, and then incubated with TUNEL reaction mixture for 60 min at 37°C. After washing with 2×SSC, slides were immersed in PBS with 1 μg/ml Propidium Iodide (PI) for 5 min in the dark and then washed with PBS. Fluorescence labeling was visualized and photographed (100× magnification) with a fluorescence microscope and with a digital camera. For TUNEL staining of the tumor xenografts, 4-μm tissue sections of tumor xenografts from in vivo experiments were stained using the ApopTag Peroxidase in Situ Oligo Ligation Apoptosis Detection Kit according to the manufacturer's protocol.

Xenografts in nude mice[1]
Nude mice (ATHYMIC NU/NU; HSD) at age 4–6 weeks with a body weight of 18–25 g were used for the experiments. Mice were injected subcutaneously at the dorsal region with 1 × 107 viable HepG2 cells. After 5 weeks, when tumors reached approximately 0.4–0.5 cm in diameter, mice were randomized into groups (n = 5) to be intratumorally injected with Toxoflavin (Xanthothricin; Toxoflavine; PKF-118-310) , PKF115-584 or CGP049090 (each diluted in PBS) at the dose of 1 mg/kg twice a week. Control mice (n = 5) were injected with PBS. Tumor size was measured with digital calipers every 3 days and was calculated using the formula π/6 × larger diameter × [smaller diameter].

Oral LD50 in mice: 8400 ug/kg. Kidneys, ureters, and bladder: hematuria; Gastrointestinal tract: hypermotility, diarrhea; Sensory organs and special senses: lacrimation; eyes. Antibiotics and Chemotherapy, 4(259), 1954. Intraperitoneal injection of LD50 in mice: 3 mg/kg. Journal of Organic Chemistry, 31(900), 1966 [PMID:5907865] Intravenous injection of LD50 in mice: 1700 ug/kg. Sensory organs and special senses: lacrimation; eyes; Gastrointestinal tract: hypermotility, diarrhea; Kidneys, ureters, and bladder: hematuria. Antibiotics and Chemotherapy, 4(259), 1954.

[1]. Small molecule antagonists of Tcf4/beta-catenin complex inhibit the growth of HCC cells in vitro and in vivo. Int J Cancer. 2010 May 15;126(10):2426-36.

[2]. Identification and characterization of PKF118-310 as a KDM4A inhibitor. Epigenetics. 2017 Mar 4;12(3):198-205.

Toxoflavin is a pyrimidotriazine compound with the chemical name 1,6-dimethyl-1,5,6,7-tetrahydropyrimido[5,4-e][1,2,4]triazine, containing carbonyl groups at positions 5 and 7. It possesses multiple functions, including antitumor activity, toxic activity, Wnt signaling pathway inhibitory activity, apoptosis-inducing activity, bacterial metabolite activity, antibacterial activity, and virulence factor activity. It is a pyrimidotriazine compound and also a carbonyl compound. Its function is similar to fulvic acid. Toxoflavin has been reported to be detected in Streptomyces hiroshimensis and Burkholderia glumae, with relevant data available. Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Due to its inherent resistance to standard chemotherapy, the development of novel selective chemotherapeutic drugs is imperative. The Wnt/β-catenin signaling pathway plays a crucial role in development and tumorigenesis and is aberrantly activated in hepatocellular carcinoma (HCC). This study aimed to evaluate the in vitro and in vivo activity of three small molecule antagonists of the Tcf4/β-catenin complex (PKF118-310, PKF115-584, and CGP049090) against HCC cell lines. In vitro experiments showed that all three compounds exhibited dose-dependent cytotoxicity against three HCC cell lines (HepG2, Hep40, and Huh7), but their cytotoxicity against normal hepatocytes (derived from three donors) was at least 10-fold lower, as determined by ATP activity assays. In HepG2 and Huh7 cells, antagonist treatment reduced Tcf4/β-catenin binding capacity and transcriptional activity, accompanied by downregulation of the expression of endogenous Tcf4/β-catenin target genes c-Myc, cyclin D1, and survivin. In HepG2 and Huh7 cells, antagonist treatment induced apoptosis and G1/S phase cell cycle arrest. All antagonists inhibited in vivo tumor growth in the HepG2 xenograft model, accompanied by apoptosis and decreased expression of c-Myc, cyclin D1, and survivin. Our results suggest that these three Tcf4/β-catenin complex antagonists are potential chemotherapeutic agents that may provide a pathway-specific option for the clinical treatment of hepatocellular carcinoma (HCC). [1]

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Epigenetic modifications play an important role in the regulation of gene expression. In particular, histone post-translational modifications play a key role in the organization of functional chromatin. A number of drugs have been found that can inhibit or activate certain protein families involved in epigenetic histone regulation, some of which have been approved by the FDA for the treatment of cutaneous T-cell lymphoma or are undergoing phase I/II/III clinical trials for solid tumors. Although some protein families, such as histone deacetylases and their inhibitors, have been well studied, our understanding of histone lysine demethylases remains incomplete. We describe the computer simulation, in vitro and cellular characterization of compound PKF118-310, a transcription factor 4 (TCF4)/β-catenin signaling pathway antagonist and also a KDM4A inhibitor. The potential inhibitory activity of PKF118-310 was discovered through virtual screening of KDM4A crystal structures. Our self-developed peptide-based histone trimethylation assay confirmed its potent inhibitory activity against KDM4A. Its protein targets were identified by cellular thermal displacement analysis. The anticancer activity of PKF118-310 was observed in both liquid tumor cells and solid tumor cells, showing a dose- and time-dependent effect. We confirmed the inhibitory effect of PKF118-310 on KDM4A, an effect that has not been previously reported. Our results provide a possibility for the development of the first KDM4A-specific inhibitor and derivative. [2]

C7H7N5O2

193.166

193.06

C, 43.53; H, 3.65; N, 36.26; O, 16.57

84-82-2

Toxoflavin-13C4;2300178-70-3

66541

Light yellow to yellow solid powder

1.65g/cm3

276.7ºC at 760 mmHg

121.1ºC

1.76

-0.7

0

3

0

14

408

0

SLGRAIAQIAUZAQ-UHFFFAOYSA-N

InChI=1S/C7H7N5O2/c1-11-6(13)4-5(10-7(11)14)12(2)9-3-8-4/h3H,1-2H3

1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7-dione

PKF118310; PKF118 310; Toxoflavin; 84-82-2; Xanthothricin; Toxoflavine; Xanthotricin; PKF-118-310; 1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7-dione; GNF-Pf-67; PKF118-310

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~129.43 mM)
H2O : ~10 mg/mL (~51.77 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (12.94 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (12.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (517.71 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)