Apoptosis In Cells Of Hepatoma And Prostate Cancer Induced By Diterpenoid B Derived From Rabdosia Excise

Objective: The genus of Rabdosia is distributed mainly in China. Anumber of diterpenes have been founded from Rabdosia plants.Among these are ent-kaurene diterpenes having antibacterial, anti-inflammatory and tumor inhibiting activities, etc. Systemic study ofthese plants is helpful to the exploitation of novel antitumor drugsfrom natural source. Diterpenoid B (DB) was isolated from Rabdosiaexcise, and until now, biological activities of DB was not yet clear inthe world, so the aim of this paper is to determine the in vivo and invitro effect of DB on the cell line of hepatoma (SMMC-7721 and H22)and prostatic cancer (PC-3) and its related mechanisms.Methods: cell line in culture medium in vitro was treated withdifferent concentrations of DB, and as a reductant, NAC was selectedto antagonize the activities of DB. MTT assay was used to detect thecell growth inhibitory rate, and morphologic changes of cells wasobserved by phase contrast microscope, Apoptosis of DB treated cellline was confirmed by the assay of acridine orange and ethidiumbromide staining and the detection of chromosomal DNAfragmentation with the classic DNA ladder method. And intracellularreactive oxygen species (ROS) level and expression of GSH-Px andCAT mRNA was analyzed by DCFH-DA fluorospectrophotometryand RT-PCR methods respectively. Meanwhile, tumor inhibitingactivity of DB in vivo was evaluated with model of tumor-bearingmice.Results: DB treated cells showed the retarded proliferation and lessdivision, suspension cell and collapse cell also appeared. A typicalmorphological change of apoptosis including condensation ofchromatin and nuclear fragmentation was observed. And acharacteristic ladder of DNA fragments also indicated DNA disruptionand apoptosis by agarose gel electrophoresis.The cell growth inhibitory rate was assayed using the microculturetetrazolium method. The results showed that various dosage of DBcaused the inhibition of cell proliferation, the growth inhibitory rate of2, 4, 8, 16, 32μg/ml DB on SMMC-7721 cell was 89.373%, 73.833%,54.515%, 50.769% and 46.081% respectively, and the growthinhibitory rate of 0.5, 1, 2, 4, 8, 16, 32μg/ml DB on PC-3 cell was84.164%, 82.83%, 81.312%, 75.338%, 52.192%, 40.399% and39.324% respectively. The number of viable cells was graduallydecreased following the increase of DB treated time. DB could inducethe apoptosis of SMMC-7721 and PC-3 cell in a dose-and time-dependent manner. DB also inhibited the growth of implantationtumor of H22 in vivo, inhibitory rate of 1, 3 and 10mg/kg DB was10.05%, 45.67% and 70.14% respectively. DB induced the elevatedintracellular ROS level, and the lower expression of GSH-Px andCAT mRNA in SMMC-7721 and PC-3 cell.NAC could reverse the change of various factors induced by DB, andantagonize the apoptosis of tumor cells.Discussion: It had been proven that DB had an antitumor activity viaits cytotoxic and apoptosis inducing effects on tumor cells in thisstudy. The mechanism of DB anti-proliferation is not clear now, but itcan be postulated that DB inhibit the proliferation of tumor cell bydown-regulation of telomerase activity, and inhibition ofangiogenesis in vivo. Antitumor activity of DB was also related withits apoptosis inducing activity by interfering redox balance, andresulting in the enhanced oxidizing and weaken reduction capability.This result was supported by some experiments of diterpenesinduced apoptosis. It was reported that apoptosis inducing protein(AIF) and arsenic trioxide could induced the increase of ROS andDNA fragmentation, and this could be reversed by NAC and CAT. Inthis study, it potentially proved apoptosis of tumor cells induced withDB via oxidative stress pathway that DB increased the intracellularROS level and NAC could antagonize DB induced apoptosis.Conclusion: DB, an active component of Rabdosia excise, couldexert its antiproliferation and apoptosis inducing function. One ofantitumor mechanisms of DB is that it triggered the apoptosis oftumor cells through oxidative stress pathway. The finding ofantitumor activity of DB outlined the prospect of expoitation ofRabdosia excise.