Marine natural products and related compounds in clinical and advanced preclinical trials. prostate carcinoma and bladder carcinoma cell lines after 72 h of treatment. The values are shown in Table ?Table1.1. C, Percentage of alive cells after treatment 7-Methylguanine with 1 M of MonA, determined by the trypan blue based viability assay. D, IC50 of MonA determined by the MTT assay. E, Trypan blue based viability assay. NCCIT and NCCIT-R cells were treated with MonA for 48 h. Time- and dose-dependent effects of MonA on NCCIT-R cells were examined by trypan blue based viability assay. n.s.: not significant. F, Effect of MonA in combination with cisplatin on NCCIT-R cells, examined by the MTT assay. Cells were co-treated with different concentrations of the single substances or their 7-Methylguanine combination for 48 h at a constant molar ratio. The combinational index (CI) values were calculated with CompuSyn software. The ratio of the substances is usually C(MonA) : C(cisplatin) = 1.2 : 10. In this study, we characterize the cytotoxic efficacy and the mode of action of this marine compound in human genitourinary malignancy cell lines with defined levels of resistance against classical anti-tumor treatments such as androgen-deprivation, docetaxel, or cisplatin. RESULTS MonA is usually more active against malignancy cells than against non-malignant cells Cytotoxic activity of MonA (Fig. ?(Fig.1A)1A) was evaluated in human malignancy cells and non-malignant human cells by MTT assay and trypan blue assay. Amazingly, GCT, prostate malignancy, and bladder malignancy cell lines were found to be equally and highly sensitive to MonA (including androgen-independent PC3 and DU145 cells), while non-malignant cells were affected to a lower lengthen (Fig. 1B, 1C; Table ?Table11). Table 1 IC50 of MonA and cisplatin in non-malignant cell lines and urogenital malignancy cell lines after 72 h of treatment decided with MTT assay as previously explained . Anisomycin, docetaxel (10 mg/ml) and cisplatin (cis-diamminedichloroplatinum (II), 1 mg/ml) were purchased from NeoCorp (Weilheim, Germany), acridine orange and calpeptin from Sigma (Taufkirchen, Germany), MG-132 from Calbiochem (Darmstadt, Germany), NH4Cl and Coomassie amazing blue G 250 from Carl Roth (Karlsruhe, Germany), 3-methyladenine and z-VAD(OMe)-fmk (referred here as z-VAD-fmk) from Enzo Life Sciences (Farmingdale, NY, 7-Methylguanine USA), leupeptin from Serva (Heidelberg, Germany), protease inhibitors cocktail (total Mini EDTA-free) from Roche (Munnheim, Germany). Main and secondary antibodies used are outlined in the supplementary. Cell lines and culture conditions The human prostate malignancy cell lines PC3 (docetaxel resistant, androgen-independent), DU145 (docetaxel sensitive, androgen-independent), LNCaP (docetaxel sensitive, androgen-dependent) [41, 42], human bladder malignancy cell lines RT112, RT4, 486p, T24, human embryonic kidney cell collection HEK 293T, human embilical vascular endothelium cell collection HUVEC, as well as human fibroblast cell lines MRC-5 and MRC-9 were obtained from ATCC (Manassas, VA, USA). The human germ cell tumor malignancy cell collection NCCIT was obtained from DSMZ (Braunschweig, Germany). TCam-2 and 2102EP cells were kindly provided by Prof. L. Looijenga (Rotterdam, The Netherlands). The cisplatin-resistant sublines NCCIT-R and 2102EP-R have been generated as reported before [16, 17]. Cells were cultured according to the manufacturers instructions (culture conditions are explained in the supplementary). Cells were constantly kept in culture for a maximum of 3 months, and were routinely inspected microscopically for stable phenotype and regularly checked for contamination with mycoplasma. Cell collection authentication was performed by DSMZ (Braunschweig, Germany) using highly polymorphic short tandem repeat loci. MTT-based drug sensitivity assay The cytotoxicity of individual substances and drug combinations was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Mouse monoclonal to BDH1 assay, which was performed as previously explained . Examination of synergistic/antagonistic effect of drug combination Determination of synergistic or antagonistic drug effects in combination assays was performed using the Chou-Talalay method . Data were generated by MTT assay. The combinational index (CI) was calculated for the constant drugs ratio with the CompuSyn v.1.0. software (ComboSyn, Inc., Paramus, NJ, USA). Synergism is usually defined as a CI < 1, whereas antagonism is usually defined by a CI > 1. The MTT assay was used to examine the combination of MonA at the IC50 with defined inhibitors of autophagy or LMP, or with the IC50 of cisplatin. Doses of the drugs utilized for combination treatment are shown in the supplementary (Table S3). All experiments were performed in triplicates and were repeated at least three times. trypan blue-based viability assay The effect of MonA on cell viability was evaluated by trypan blue exclusion assay using semi-automated cell count with a Beckman Coulter Vi-CELL (Beckman Coulter, Krefeld, Germany) as explained before . Protein preparation and western blotting Preparation of protein.