In Vitro Study On Antitumor Activity Mechanism Of Docetaxel Derivative 10j Towards Human Prostate Cancer Cells(PC-3)

Paclitaxel,one of the most important anticancer drug in the world currently,was originally discovered from the bark of Taxus brevifolia Nutt.by Wall and Wani in 1967.It is currently used for the treatment of kinds of cancers,such as breast,lung,liver and neck cancers and so on[8-11].Docetaxel is paclitaxel derivative,and its anticancer spectrum and activity were higher than paclitaxel,therefore synthesis of novel docetaxel derivatives has been international hotspots issues.This paper studies the antitumor mechanism of docetaxel derivative lOj which was synthesized by our group,providing an important pharmacological basis for new drug development.Smac/DIABLO is also an important molecule that regulates the function of IAPs.The Smac/DIABLO protein resides in the mitochondria of healthy cells,and is released upon apoptotic stress with similar kinetics to cytochrome c[32,33].Smac/DIABLO can bind to the BIR domain of IAPs,thereby interfering with either caspase-3/-7 or caspase-9 inhibition.Here,we report for the first time that the interaction between Smac/DIABLO and Survivin is an important step for suppressing the anti-apoptotic function of Survivin in compound 10j-induced apoptosis.The results were as follows:1.Growth inhibition was analyzed by MTT assay.It was found that docetaxel derivative 10j markedly inhibited proliferation of several tumor cell lines,including human colon cancer cells(HT-29,IC50:118.30±1.55 nM),human prostate cancer cells(PC-3,IC50:91.38±2.94 nM),human lung adenocarcinoma cells(A549,IC50:117.02±1.14 nM),human cervical cancer cells(Hela,IC50:129.59±1.87 nM)and human breast cancer cells(MCF-7,IC50:137.38±1.79 nM),suggesting that PC-3 cell line is the most sensitive.The IC50 of docetaxel derivative 10j towards African green monkey kidney(Vero)cells were 54.26±3.52 ?M.2.Hoechst 33258 staining showed that there were considerable morphological changes in the nuclear chromatin.Control PC-3 cells that were not subjected to docetaxel derivative 10j treatment did not exhibit chromatin condensation.The nuclei were stained in a less bright blue and the color was homogeneous.By contrast,after treatment with docetaxel derivative 10j for 48 h,we found that most of the cells exhibited very intense staining of condensed and fragmented chromatin,and some of them formed typical apoptotic bodies.Only a few nuclei still displayed normal morphology.These data clearly indicate that docetaxel derivative 10j induces apoptosis in PC-3 cells.3.Apoptotic population of PC-3 cells was analyzed by flow cytometry using annexinV-FITC/PI.The apoptosis results showed that the apoptotic population increased in a concentration-dependent manner after the cells treated with docetaxel derivative 10j.4.The DNA content was measured by flow cytometry.As shown in concentration kinetic measurements,exposure to docetaxel derivative 10j in a range from 0 to 60 nM caused an deincrease of the G2/M phase population.Hence,docetaxel derivative 10j exerted growth-inhibitory effects via G2/M phase arrest in a concentration-dependent manner.5.We investigated docetaxel derivative 10j induce alterations in mitochondrial membrane potential of PC-3 cells by flow cytometry.Flow cytometry analysis showed that the percentage of cells in low fluorescence produced a time-dependent increase,indicating that docetaxel derivative 10j caused depolarization of ??m.These data showed that docetaxel derivative 10j induces apoptosis accompanied by the alterationsin the mitochondrial membrane potential.6.The accumulation of G2/M cells and the intensity of mitotic arrest after docetaxel derivative 10j exposure were confirmed biochemically by changes of cyclin B1 and ?-tubulin,respectively.The changes seen in Survivin protein were almost identical to ?-tubulin.During these changes,we noted that Smac/DIABLO proteins were also increased concurrently with Survivin.Meanwhile,our results showed that a very strong immunoreactivity for Smac/DIABLO was detected in the Survivin immunoprecipitant,implying that Smac binds to Survivin in the nucleus.Meanwhile,docetaxel derivative 10j inhibited the Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release.Cytochrome c released from mitochondria is associated with the activation of caspase-3 and caspase-9 cascade.All signal transduction pathway played a role in initiating apoptosis.To the best of our knowledge,the cytotoxic activity of docetaxel derivative 10j towards cancer cells was studied for the first time in the present study.Our studies encourage the development of novel and efficient therapeutic approaches for cancer.