Acalypha wilkesiana extracts induce apoptosis by causing single strand and double strand DNA breaks.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Acalypha wilkesiana have been used empirically by traditional healers in Southwest Nigeria together with other plants as a powder mixture to treat patients with breast tumours and inflammation. AIM OF THE STUDY: There is an increasing interest among researchers in searching for new anticancer drugs from natural resources, particularly plants. This study aimed to investigate the anticancer properties of Acalypha wilkesiana extracts and the characteristics of DNA damage against brain and lung cancer cells. MATERIALS AND METHODS: The antiproliferative activity of Acalypha wilkesiana extracts (ethyl acetate, hexane, and ethanol) was examined on human glioma (U87MG), human lung carcinoma (A549), and human lung fibroblast (MRC5) cells. RESULTS: Cell viability MTT assay revealed that ethyl acetate extract of the plant possessed significant antiproliferative effects against both U87MG (GI(50)=28.03 ± 6.44 µg/ml) and A549 (GI(50)=89.63 ± 2.12 µg/ml) cells (p value<0.0001). The hexane extract was found to exhibit crucial antiproliferative effects on U87MG (GI(50)=166.30 ± 30.50 µg/ml) (p value<0.0001) but not on A549 cells. Neither plant extract possessed noticeable antiproliferative effects on the non-cancerous MRC5 cells (GI(50)>300 µg/ml). The ethanol extract showed no antiproliferative effects on any cell line examined. Haematoxylin & Eosin (H & E) staining and single cell gel electrophoresis (SCGE) comet assay confirmed that plant extract-treated cells underwent apoptosis and not necrosis. SCGE comet assays confirmed that plant extracts caused both single strand (SSB) and double strand (DSB) DNA breaks that led to the execution of apoptosis. CONCLUSION: The extracts (especially ethyl acetate and hexane) of Acalypha wilkesiana possess valuable cytotoxic effects that trigger apoptosis in U87MG and A549 cancer cells through induction of DNA SSBs and DSBs.

Preclinical toxicokinetic evaluation of phortress [2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole lysylamide dihydrochloride] in two rodent species.

BACKGROUND AND AIMS: The 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole prodrug Phortress exerts potent and selective antitumour activity in vitro and in vivo. Preclinical toxicokinetic studies in 2 rodent species were undertaken to determine Phortress' maximum tolerated dose and advise a safe starting dose for clinical evaluation. METHODS: Plasma pharmacokinetic parameters were determined by high-performance liquid chromatography and fluorescence detection following Phortress administration to mice (10 mg/kg, intravenously on days 1 and 8). Phortress (20 mg/kg, on days 1 and 8) was administered to CYP1A1/betaGAL reporter mice; tissues were examined macro- and microscopically. Toxicological and pharmacodynamic endpoints were examined in organs of rodents receiving Phortress (10 mg/kg or 20 mg/kg, on days 1 and 8). CYP1A1 expression and Phortress-derived DNA adducts were determined in lungs and livers (on days 11 and 36). RESULTS: No accumulation of Phortress was detected in murine plasma. beta-Galactosidase activity inferred Phortress-derived induction of cyp1a1 transcription in the livers of transgenic mice; no total body weight loss was encountered in these animals. However, a fall in lung:body weight and kidney:body weight ratios, raised serum alkaline phosphatase levels and hepatic histopathological disturbances in animals receiving 20 mg/kg Phortress indicate organ sites of potential toxicity. CYP1A1 protein was induced transiently in the lungs of both species and in the livers of rats. Elimination of hepatic DNA adducts and rat pulmonary adducts was evident; however, murine pulmonary adducts persisted. CONCLUSION: Rodent preclinical toxicology established that mice represent the more sensitive rodent species, resolving a maximum tolerated dose of 10 mg/kg Phortress.

In vitro evaluation of amino acid prodrugs of novel antitumour 2-(4-amino-3-methylphenyl)benzothiazoles.

Novel 2-(4-aminophenyl)benzothiazoles possess highly selective, potent antitumour properties in vitro and in vivo. They induce and are biotransformed by cytochrome P450 (CYP) 1A1 to putative active as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. The lipophilicity of these compounds presents limitations for drug formulation and bioavailability. To overcome this problem, water soluble prodrugs have been synthesised by conjugation of alanyl- and lysyl-amide hydrochloride salts to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles. The prodrugs retain selectivity with significant in vitro growth inhibitory potency against the same sensitive cell lines as their parent amine, but are inactive against cell lines inherently resistant to 2-(4-aminophenyl)benzothiazoles. Alanyl and lysyl prodrugs rapidly and quantitatively revert to their parent amine in sensitive and insensitive cell lines in vitro. Liberated parent compounds are sequestered and metabolised by sensitive cells only; similarly, CYP1A1 activity and protein expression are selectively induced in sensitive carcinoma cells. Amino acid prodrugs meet the criteria of aqueous solubility, chemical stability and quantitative reversion to parent molecule, and thus are suitable for in vivo preclinical evaluation.

The effect of fetal calf serum on growth arrest caused by activators of protein kinase C.

The growth of human-derived A549 lung carcinoma cells is inhibited by activators of protein kinase C (PKC) such as 12-O-tetradecanoylphorbol- 13-acetate (TPA). In this study, the effect of serum deprivation on TPA-induced growth retardation has been investigated. Cells cultured with 10% FCS and TPA (10(-8) M) stopped growing for 6 days, whereas inhibition of DNA synthesis caused by TPA in cells which were grown in medium containing the serum substitute ultraser lasted for less than 48 hr. The ability of cells to respond to the growth-inhibitory potential of TPA decreased with decreasing amounts of FCS in the cellular medium. Addition of fetuin or epidermal growth factor (EGF) to incubates with serum-deprived cells increased the ability of TPA to affect growth, but addition of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) or retinoic acid (RA) was without effect. Growth arrest caused by bryostatin I, another PKC activator, was equally transitory in serum-supplemented and serum-deprived cells. Cytosol of serum-deprived cells contained only 32% of specific phorbol ester binding sites compared to cells grown with FCS; PKC enzyme activity and immunodectable protein were similarly reduced in cells grown without FCS. There was no difference in rate of TPA-induced down-regulation of PKC activity and cytosolic phorbol ester receptor sites between cells grown with or without serum.