harmine and thiazolyl-blue

The effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Harman and norharman at a concentration of 20 microM and 100 microM showed 49.4% and 49.5% inhibition of dopamine content for 48 h, respectively. The IC50 values of harman and norharman were 21.2 microM and 103.3 microM. Dopamine content, tyrosine hydroxylase (TH) activity and TH mRNA levels were decreased during the first 6 h, maintained for up to 48 h and then gradually recovered at 72 h after exposure to 20 microM harman and 100 microM norharman. Under the same conditions, the intracellular cyclic AMP levels and Ca2+ concentrations were also decreased by harman and norharman. In addition, harman and norharman at concentrations higher than 80 microM and 150 microM caused cytotoxicity at 48 h in PC12 cells. Non-cytotoxic ranges of 10-30 microM harman and 50-150 microM norharman inhibited L-DOPA (20-50 microM)-induced increases in dopamine content at 48 h. Harman at 20-150 microM and norharman at 100-300 microM also enhanced L-DOPA (20-100 microM)-induced cytotoxicity at 48 h with an apoptotic process. These results suggest that harman and norharman inhibit dopamine biosynthesis by reducing TH activity and enhance L-DOPA-induced cytotoxicity in PC12 cells.

Topics: Animals; Antiparkinson Agents; Apoptosis; Aromatic-L-Amino-Acid Decarboxylases; Blotting, Northern; Calcium; Carbolines; Cell Survival; Cyclic AMP; Dopamine; Flow Cytometry; Harmine; In Situ Nick-End Labeling; Levodopa; Membrane Potentials; Mitochondrial Membranes; PC12 Cells; Rats; RNA; Tetrazolium Salts; Thiazoles; Tyrosine 3-Monooxygenase

Beta-carboline alkaloids such as harmine are present in medicinal plants such as Peganum harmala that have been used as folk medicine in anticancer therapy. In our study, 9 harmine derivatives (including harmine) were investigated for their antitumor effects and acute toxicities in mice, and the structure-activity relationship (SAR) was also analyzed. Administration of these compounds resulted in tumor inhibition rates of 15.3-49.5% in mice bearing Lewis Lung Cancer, sarcoma180 or HepA tumor, with the highest value of 49.5% from compound 6. Acute toxicity studies showed that all these compounds except compounds 2 and 5 caused remarkable acute neurotoxicities manifested by tremble, twitch and jumping. SAR analysis indicated that the formate substitution at R3 of the tricyclic skeleton reduced their neurotoxicity, while the short alkyl or aryl substitution at R9 increased the antitumor activity. The harmine and its derivatives resulted in in vitro cytotoxicity (IC50) values of 0.011-0.021 micromol/ml in HepG2 cells, with compound 8 being the most potent among all agents tested. Compounds 1, 6, 7 and 8 induced apoptosis in HepG2 cells, with the highest apoptotic rate (55.34%) from compound 6. Western blotting analysis demonstrated that compound 6 completely inhibited the expression of Bcl-2 gene, and compounds 1 and 8 produced a significant inhibition by 40 and 60%, respectively, compared to the control, while compound 7 did not alter the level of Bcl-2. Compounds 1, 6, 7 and 8 upregulated the expression of death receptor Fas by approximately 50-120%. All these findings indicate that compounds with both substitutions at R3 and R9 (such as compound 5) have high antitumor activity and low toxicity, which might be chosen as lead molecules for further development. Further studies on the effects of harmine derivatives on key regulators for tumor cell apoptosis are needed.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Lewis Lung; Cell Line; Cell Line, Tumor; Cell Proliferation; Coloring Agents; DNA; fas Receptor; Flow Cytometry; Harmine; Humans; Immunoblotting; Inhibitory Concentration 50; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Models, Chemical; Monoamine Oxidase Inhibitors; Neoplasm Transplantation; Peganum; Proto-Oncogene Proteins c-bcl-2; Sarcoma; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Suppressor Protein p53; Up-Regulation