Acetylshikonin isolated from Lithospermum erythrorhizon roots inhibits dihydrofolate reductase and hampers autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice.

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.

A multilayer screening approach toward the discovery of novel Pf-DHFR inhibitors.

A small yet diverse xanthone library was build and computationally docked against wild type Pf-DHFR by Molegro Virtual Docker (MolDock). For analysis of results an integrated approach based on re-ranking, scaling (based on heavy atom counts), pose clustering and visual inspection was implemented. Standard methods such as self-docking (for docking), EF analysis, average rank determinations (for size normalization), and cluster quality indices (for pose clustering) were used for validation of results. Three compounds X5, X113A and X164B displayed contact footprints similar to the known inhibitors with good scores. Finally, 16 compounds were extracted from ZINC data base by similarity based screening, docking score and drug/lead likeness. Out of these 16 compounds, 11 displayed very close contact footprints to experimentally known inhibitors, indicating there potential utility in further drug discovery efforts.

Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening.

BACKGROUND: Plasmodium vivax is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous in vitro culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites. METHODS: Plasmodium falciparum and Plasmodium berghei parasites were transfected with DNA constructs bearing P. vivax dhfr-ts pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous dhfr-ts of P. falciparum and P. berghei parasites with P. vivax homologous genes. The integration of Pvdhfr-ts genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays. RESULTS: Transgenic P. falciparum and P. berghei lines stably expressing PvDHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type PvDHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant PvDHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites. CONCLUSION: With the permanent integration of Pvdhfr-ts gene in the genome, the transgenic Plasmodium lines expressing PvDHFR-TS are genetically stable and will be useful for screening anti-P. vivax compounds targeting PvDHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.

Greeen tea extracts lower serum folates in rats at very high dietary concentrations only and do not affect plasma folates in a human pilot study.

Green tea catechins (GTC) have been shown to inhibit the activities of enzymes involved in folate uptake. Hence, regular green tea drinkers may be at risk of impaired folate status. The present experiments aimed at studying the impact of dietary GTC on folate concentrations and metabolism. In a human pilot study (parallel design) healthy men consumed for 3 weeks 6 capsules (approximately 670 mg GTC) per day (2 capsules with each principal meal) containing aqueous extracts of the leaves of Camellia sinensis (n=17) or placebo (n=16). No differences in plasma folate concentrations were observed between treatments. We further fed groups of 10 male rats diets fortified with 0, 0.05, 0.5, 1, or 5 g GTC/kg for 6 weeks. Only at the highest intake, GTC significantly decreased serum 5-methyl-tetrahydrofolate concentrations in rats, while mRNA concentrations of reduced folate carrier, proton-coupled folate transporter/heme carrier protein 1, and dihydrofolate reductase (DHFR) remained unchanged in intestinal mucosa. Using an in vitro enzyme activity assay, we observed a time- and dose-dependent inhibition of DHFR activity by epigallocatechin gallate and a green tea extract. Our data suggest that regular green tea consumption is unlikely to impair folate status in healthy males, despite the DHFR inhibitory activity of GTC.

Dihydrofolate reductase and cell growth activity inhibition by the beta-carboline-benzoquinolizidine plant alkaloid deoxytubulosine from Alangium lamarckii: its potential as an antimicrobial and anticancer agent.

Beta-carboline-benzoquinolizidine plant alkaloid deoxytubulosine (DTB) was evaluated and assessed for the first time for its biochemical and biological activity employing the biomarker dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) as the probe enzyme, a key target in cancer chemotherapy. DHFR, employed in the present investigations was purified from Lactobacillus leichmannii. DTB, isolated from the Indian medicinal plant Alangium lamarckii was demonstrated to exhibit potent cytotoxicity. The alkaloid potently inhibited the cell growth of L. leichmannii and the cellular enzyme activity of DHFR (IC50=40 and 30 microM for the cell growth and enzyme inhibitions, respectively). DTB concentrations >75 microM resulted in a total loss of the DHFR activity, thus suggesting that the beta-carboline-benzoquinolizidine plant alkaloid is a promising potential antitumor agent. Our results are also suggestive of its potential antimicrobial activity. DTB binding to DHFR appears to be slow and reversible. Inhibition kinetics revealed that DHFR has a Ki value of 5x10(-6) M for DTB and that the enzyme inhibition is a simple linear 'non-competitive' type.

Renal and hepatic toxicity after high-dose 7-hydroxymethotrexate in the rat.

To examine directly the hepatic and renal toxicity of 7-hydroxymethotrexate (7-OH-MTX) without interference of the parent compound methotrexate (MTX), we purified and gave 100 mg/kg 7-OH-MTX to rats, a dose resulting in serum levels of 7-OH-MTX comparable with those achieved in the clinic after the administration of high-dose MTX (HD-MTX). After only 5 h, the 7-OH-MTX-treated rats demonstrated 2.6-fold increases in serum creatinine values and 2-fold elevations in serum aspartate aminotransferase (ASAT) levels as compared with the controls. Morphologic evidence of toxicity, however, was apparent only in the kidneys. Intraluminal cellular debris containing membranous material and deteriorated organelles was seen, but no precipitate of the delivered drug. The peak serum concentration of 7-OH was up to 939 microM, and concentrations of 7-OH-MTX declined triphasically, showing a t1/2 alpha value of 2.45 min, a t1/2 beta value of 30.5 min, and a terminal half-life (t1/2 gamma) of 240 min. The total clearance value was 14.5 ml min-1 kg, and the postdistributional volume of distribution (V beta) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.

Screening for new antifolates of microbial origin and a new antifolate AM-8402.

A screening method was established for new specific inhibitors of folate metabolism. Culture broths of soil isolates were selected based on relative microbial activity. A culture, to be retained, must be active against Enterococcus faecium grown in a medium which contains a limited amount of pteroic acid but lacks activity against the microorganism grown in a medium supplemented with thymidine. By this screening method, three new antibiotics, diazaquinomycins A and B and AM-8402 were selected from eight thousand soil isolates. The isolation and structures of diazaquinomycins have been reported. AM-8402 is a new antifolate active against Gram-positive bacteria and mycoplasmas. It consists of a nanaomycin D moiety as chromophore and a deoxysugar and is structurally related to medermycin.