Characterization and bioactivity of cyclotides from Psychotria leptothyrsa (Rubiaceae)
Description
Cyclotides, circular peptides isolated from plants of the Violaceae and Rubiaceae families, have a unique cyclic cystine knot topology and may play a role in plant defense. In addition to being antibacterial and insecticidal, cyclotides also display antitumor and anti-HIV activities. Their mechanism of bioactivity is not fully understood, but they disrupt cellular function by forming pores on lipid membranes. Reports on the distribution of cyclotides in Rubiaceae are limited; this dissertation focuses on the discovery, characterization and bioactivity of cyclotides in Psychotria (Rubiaceae). Eleven species of Psychotria were screened for cyclotides; six novel cyclotides (named psyle A - psyle F) were purified and sequenced from extracts of Psychotria leptothyrsa var. longicarpa using a combination of liquid chromatography-mass spectrometry and tandem nanospray MS-MS sequencing. The bioactivity of the psyle cyclotides, as well as the cyclotide cycloviolacin O2 (CyO2) from Viola odorata was examined using bioassays (fluorometric microculture, cell proliferation, SYTOX Green nucleic acid stain, trypan blue and saquinavir uptake) and fluorescence microscopy. Cytotoxic, chemosensitizing and pore-forming abilities of cyclotides were monitored in the human adenocarcinoma breast cancer cell line (MCF-7), its drug resistant sub-line (MCF-7/ADR), an uninfected T-lymphocytic cell line (HuT78) and its HIV-infected sub-line (HTLVIIIB), and the human lymphoma cell line (U-937GTB). Cyclotides displayed potent, dose-dependent cytotoxicities (IC50 = 0.72--26 muM), and co-exposure to cyclotides significantly enhanced doxorubicin-induced toxicity (IC50 = 0.39--0.76 muM). Cyclotides formed pores on membranes of breast cancer cells, and this pore-formation correlated with their chemosensitizing abilities. Psyle E and CyO2 were the most potent of the cyclotides tested, but a linear rubiaceous cyclotide (psyle C) also maintained cytotoxicity (IC50 = 0.47--8.70 muM). CyO2 formed rapid (< 10 minutes) and stable (24 hours) pores on cellular membranes, enhanced the uptake of saquinavir in T-lymphocytes (HuT78) by more than three-fold, and was five times more potent in HIV-infected T-lymphocytes (HTLVIIIB) as compared to HuT78s. Cyclotides did not produce significant pore-formation in brain endothelial or kidney epithelial cell lines, which suggests specificity toward inducing pore-formation in highly proliferating breast tumor and HIV-infected cells. In conclusion, cyclotides may be promising candidates in targeted membrane disruption approaches to the treatment of drug resistant breast cancer and HIV