Synthesis, Optical Properties, and Antiproliferative Evaluation of NBD-Triterpene Fluorescent Probes

A fluorescent labeling protocol for hydroxylated natural compounds with promising antitumor properties has been used to synthesize 12 derivatives having fluorescent properties and biological activity. The reagent used for the synthesis of these fluorescent derivatives was 7-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl). The linkers employed to bind the NBD-Cl reagent to the natural compounds were ω-amino acids of different chain lengths. The natural triterpene compounds chosen were oleanolic and maslinic acid, as their corresponding 28-benzylated derivatives. Thus, triterpene conjugates with NBD have been studied for their optical fluorescence properties and their biological activities against cell proliferation in three cancer cell lines (B16-F10, HT-29, and HepG2), compared with three nontumor cell lines (HPF, IEC-18, and WRL68) from different tissues. The results of the fluorescence study have shown that the best fluorescent labels are those in which the ω-amino acid chain is shorter, and the carboxylic group is not benzylated. Analysis by confocal microscopy showed that these compounds were rapidly incorporated into cells in all three cancer cell lines, with these same derivatives showing the highest toxicity against the cancer cell lines tested. Then, the fluorescent labeling of these triterpene conjugates with NBD enabled their uptake and subcellular distribution to be followed to probe in detail their biological properties at the cellular and molecular level.Grupo de Investigación "Biotecnología y Química de Productos Naturales" (grupo FQM-139 del PAIDI de la Junta de Andalucía

A Diamine-PEGylated Oleanolic Acid Derivative Induced Efficient Apoptosis through a Death Receptor and Mitochondrial Apoptotic Pathway in HepG2 Human Hepatoma Cells

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Our recent studies have shown that the diamine-(PEG)ylated oleanolic acid (OADP) has strong anti-tumor effects in HCCs. In this study, we evaluated the anti-tumor mechanisms of OADP in the HepG2 HCC cell line. The cytotoxicity results showed that HepG2 cell viability was markedly reduced, with a very low 50% of cell growth inhibitory concentration (IC50, 0.14 µg/mL). We then investigated the anti-tumor mechanisms of OADP in HepG2 cells. The flow-cytometry analysis was used to evaluate cell apoptosis, indicating that 74–95% of cells were apoptotic. OADP caused cell cycle arrest in the G0/G1 phase and the loss of the mitochondrial membrane potential (MMP). Western blot analysis was performed to assess the expression levels of key proteins associated with the underlying molecular mechanism. The results showed the clear upregulation of caspase-8, caspase-9, caspase-3, Bak, p21, and p53, accompanied by the downregulation of Bcl-2. Similar results were obtained by the cotreatment with OADP and the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Agents such as OADP, which are capable of activating extrinsic and intrinsic apoptotic pathways, may represent potential HCC cancer therapies.Junta de Andalucia B1-BIO-281-UGR18 B1-FQM-217-UGR1

Synthesis and Biological Activity of Triterpene-Coumarin Conjugates

A set of 12 maslinic acid−coumarin conjugates was synthesized, with 9 being maslinic acid−diamine−coumarin conjugates at the C-28 carboxylic acid group of triterpene acid and the other three being maslinic acid−coumarin conjugates at C- 2/C-3 and/or C-28 of the triterpene skeleton. The cytotoxic effects of these 12 triterpene conjugates were evaluated in three cancer cell lines (B16-F10, HT29, and Hep G2) and compared, respectively, with three nontumor cell lines from the same or similar tissue (HPF, IEC-18, and WRL68). The most potent cytotoxic results were achieved by a conjugate with two molecules of coumarin-3-carboxylic acid coupled through the C-2 and C-3 hydroxy groups of maslinic acid. This conjugate showed submicromolar IC50 values in two of the three cancer cell lines tested (0.6, 1.1, and 0.9 μM), being between 110- and 30-fold more effective than its corresponding precursor. Furthermore, this conjugate (10) showed percentages of cell viability for the three nontumor lines of 90%. Four maslinic acid−coumarin conjugates displayed apoptotic effects in the treated cells, with total apoptosis rates of between 40 and 85%, relative to the control. Almost all the compounds assayed caused cell-cycle arrest in all cancer cell lines, increasing the number of these cells in the G0/G1 phase.Junta de Andalucia B1-FQM-217-UGR18 B1-BIO-281-UGR1