Study On Molecular Mechanisms Of Antitumor Effect Of Ent-kaurene Diterpenoids From Isodon

Plants belonging to the genus Isodon are widely used as antitumor, antibacterial, and anti-inflammatory herbs in China. Ent-kaurene diterpenoids, which are the main active constituents against tumor, are rich in the genus Isodon. Recently, it has been indicated that the ent-kaurene diterpenoids exhibited antitumor effect via induction of apoptosis in tumor cells. However, mechanisms underlying the apoptosis-inducing activity and the cellular targets of ent-kauranoids remain obscure and whether they can find clinical application still need more investigation.In this study, a series of 20-non-oxygenated-ent-kauranoids obtained from plants of Isodon were first evaluated for their inhibitory activities on the growth of human HL-60 myeloid leukemia cells.The results demonstrated that all these compounds were able to induce growth arrest in HL-60 cells. Structure-activity assay indicated that the structure ofα-methylene cyclopentanone in diterpenes is a key group for retaining the activity to induce proliferation inhibition. Melissoidesin G (MOG), a typical ent-kaurene diterpenoid amongst the compounds tested, is rich in Isodon melissoides and exhibited significant growth inhibitory effect against HL-60 cells with an IC50 of 1.98μM. The anti-proliferative activity of MOG was further determined by using a panel of 10 human cancer cell lines. Five leukemia cell lines including HL-60 displayed the most sensitivity to MOG.Next, MOG as a typical ent-kaurene diterpenoid was used to study the mechanism underlying the inhibitory effect of ent-kaurene diterpenoids on leukemic cell growth. The results showed that MOG could specifically inhibit the growth of human leukemia cell lines and primary acute myeloid leukemia (AML) blasts via induction of apoptosis, with evidences of phosphatidylserine externalization, subG1 appearance and nuclear fragmentation. Furthermore, it was shown that MOG treatment caused mitochondrialΔΨm loss and cytochrome c release associated with down-regulation of Bcl-xL and MCL-1. These results suggested that the MOG-induced apoptosis may involve the mitochondrial damage. Additionally, the initiator caspase-8 and -9 were observed to be concomitantly activated in HL-60 cells upon treatment with MOG. Collecting these data, we proposed that MOG induced caspase-dependent apoptotic death of HL-60 cells through both extrinsic and intrinsic pathways.Previous studies have suggested that redox imbalance induced by chemotherapy drugs is an important mechanism for tumor cell apoptosis. In this study, MOG rapidly decreased the intracellular glutathione (GSH) content and increased production of reactive oxygen species (ROS), resulting in the disruption of cellular redox balance. Furthermore, it was observed that thiol-containing antioxidants completely blocked MOG-induced mitochondrialΔΨm loss and subsequent cell apoptosis, while the inhibition of apoptosis by benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) only partially attenuated mitochondrialΔΨm loss, indicating that the MOG-induced redox imbalance is an early event upstream to mitochondrialΔΨm loss and caspase-3 activation.Being antioxidants, both NAC and tert-butylhydroquinone (tBHQ) pretreatments resulted in the increase of cellular GSH content in HL-60 cells, thereby significantly reducing the MOG-induced decrease of GSH levels and subsequent apoptosis. Conversely, DL-buthionine (S, R)-sulfoximine (BSO), which inhibits GSH biosynthesis, decreased the GSH levels, thus aggravating the MOG-induced GSH depletion, and eventually enhancing the apoptotic effect of MOG. All these data identified the important role of GSH depletion in the MOG-induced target cell apoptosis.Last, based on the observation that MOG treatment could cause significant intracellular GSH depletion, we proposed that MOG might enhance apoptotic effect of As₂O₃ through induction of redox imbalance. To test this hypothesis, HL-60 cells were cotreated with MOG and As₂O-3. Expectedly, it was shown that treatment of As₂O₃ in combination with MOG produced synergistic apoptotic effect in HL-60 cells and fresh AML blasts. However, this combination produced none apoptotic effect in normal human peripheral blood mononuclear cells (PBMCs). These results demonstrated that MOG treatment could render the AML cell lines more susceptible to As₂O₃-mediated apoptosis without increasing additional toxic side effect against normal cells. Taken together, the findings in our studies indicate that MOG is capable of eliciting significant growth inhibition of leukemia cells via induction of caspase-dependent apoptosis. Disruption of redox balance (mainly of GSH depletion) plays a vital role in the regulation of apoptosis induced by MOG. Furthermore, given the growth inhibitory effects of MOG against leukemia cells as well as its synergy with As₂O₃ on apoptosis of AML blasts, these findings suggest that MOG alone or administered with other anticancer agents may provide a novel therapeutic strategy for leukemia.