Study On The Structure-activity Relationship And The Anticancer Molecular Mechanism Of Diterpenoids From Labiatae Isodon Species

Nature product is a very important resource for drugs and drug lead compounds. Recent researches home and abroad show that Isodon species diterpenoids possess various bioactivities including induction of cell apoptosis, inhibition of NF-kB signaling pathway and immunity suppression. As researchers focus on those compounds which have remarkable bioactivities and incline to be developed into innovative drugs, it is necessary to unveal structure-activity relationship of all kinds of diterpenoids for investigating the mechanism of actions and intracellular targets of these compounds.This study aims at evaluating the anticancer activities of a series of natural diterpenoids in vitro and analyzing their structure-activity relationship to find out their active chemical regions. Furthermore, we investigated mechanism of action of one lead compound Melissoidesin G(MOG) ,which induced apoptosis in human myeloid leukemia HL-60 cell.The cytotoxicity of the diterpenoids was measured by MTT assay. Apoptotic cells with Annexin V/PI double staining were measured by flow cytometry. Fluorometric colorimetry and western blotting analysis were performed to measure caspases activity. The mitochondrial transmembrane potential (△(?)m) and intracellular reactive oxygen species (ROS) were measured using the fluorescent probe JC-1 and DCFH-DA. Models of tumor-bearing mice were established to observe the anticancer activity of MOG in vivo. The growth of HL-60 cells could be inhibited by these diterpenoids andα-methylene cyclopentanone in molecule structures is a key activity group which helps keeping anticancer activity of the compounds. Acetyl group or hydroxyl group in structures can affect compounds activity as well. OAc-6, OAc-11 and OH-7 enhance the anticancer activity, while OH-1, OH-2 reduce it. MOG inhibited the growth of many cell lines and induced apoptosis ,which was associated with loss of mitochondrial△Ψm, caspases activation and redox imbalance. Antioxidants NAC or GSH completely abolished MOG-induced mitochondrial△Ψm loss, caspases activation and apoptosis, while pan caspase inhibitor Z-VAD-FMK only partially attenuated mitochondrial△Ψm loss, indicating that MOG-induced redox imbalance was an early event upstream to mitochondrial△Ψm loss and caspases activation. Among the antioxidants evaluated, only NAC and GSH can completely abrogate MOG-induced cell growth inhibition, suggesting intracellular redox imbalance depends on decrease of GSH content and GSH may be the intracellular target of MOG. Abolition of the cytotoxic effects of these diterpenoids by NAC or GSH suggests that these compounds have similar anticancer mechanisms. Furthermore, MOG could inhibite tumor growth in the tumor-bearing mice , prolong their life span and improve life quality.The diterpenoid tested withα-methylene cyclopentanone all have strong anticancer activities in vitro, which suggestsα-methylene cyclopentanone is the leading active site. The study of MOG-induced HL-60 cell apoptosis indicates that MOG could inhibit the proliferation of cancer cells and induce apoptosis via disruption of redox balance, followed by perturbation of mitochondria and activation of caspase-3 and -7.