Study On Anti-growth Activity And Its Mechanism In Human Gynecologic Tumor Cell Lines By The Compounds From The Torreya Nucifer L.

Objective:Torreya nucifer L is a special kind of Taxus plants in Japan,North Korea and China, whose seeds have very strong insecticidal activity. Itis used for the treatment of tapeworm infection in China and North Korea. Asit is used as medical abortion in Japanese folk. About the studies of JapaneseTorreya chemical composition mainly focus on its leaves and heartwood. Itsroots used medicinally in the stomach, diuresis, eliminating phlegm andanthelmintic, etc. Human endometrial cancer cell line (HEC-1), humancervical cancer cell line (HeLa), human ovarian cystadenocarcinoma cell line(HAC-2), human ovarian clear cancer cell lines (HOC-21and SHIN-3) werestudied in this experiment. Three Diterpenes (18-hydroxyferruginol, kayadioland hinokiol) purified from Japanese Torreya fruit were studied in5gynecologic tumor cell lines for their proliferation inhibition in our study.Furthermore, using HEC-1cell line explore the mechanism of kayadiolinhibiting HEC-1cell proliferation.Methods:1MTT assay1.1Preparation for tumor cell lineTo modulate HEC-1tumor cell lines of the concentration of2×105个/mLwith RPMI1640complete medium.1.2Anti-tumor effect in vitro of3DiterpenesTumor cell lines (HEC-1, HeLa, HAC-2, HOC-21and SHIN-3) at thestage of logarithmic phase were inoculated in96well plates, each well50μL(containing1×104cells)and were treated for48hours with the finalconcentration0.1%DMSO as solvent control, cisplatin as positive control and3Diterpenes of final different concentration1μmol/L,10μmol/L and100μmol/L for50μL in saturated humidity,37℃and5%CO2incubator. Each group was equipped with3duplicate holes. Each hole was added5mg/mLMTT10μL before4hours to the end of cultivation. Then discarded culturesupernatant carefully, added reaction stop solution150μL, and oscillated10min to dissolve the crystallization fully. Finally the hole absorbance (OD)values was detected by Enzyme-linked immunosorbent Detector, andcalculated the cell survival rate. Experimental data was processed by TheSPSS system V13.0software, and the cell concentration-response curve of thetested drugs was fitted with the Hill model. Then the IC₅₀values of drugs wascalculated.2The analysis on situ apoptotic cell labeling and semi quantitativeHEC-1cells at the stage of logarithmic phase were inoculated in8wellChamber slide, each well100μL (containing2×10⁴cells) and with the finalconcentration0.1%DMSO as solvent control, cisplatin as positive control andkayadiol of final concentration10μmol/L for50μL in saturated humidity,37℃and5%CO2incubator12h and24h. Using terminal deoxynucleotidyltransferase (TdT enzyme) mediated by fluorescent dUTP nick end labeling(TUNEL) detection. Results according to the distribution of apoptosis positivecells. Each slice shot5positive view, each view counts to normal cell numberand the number of apoptotic cells, with average calculation of the percentageof positive cells as the cell apoptosis rate apoptotic rate, AR).3Western blottingHEC-1cells at the stage of logarithmic phase were planted in Petri dishes(containing2×10⁶cells/dish), and were treated with RPMI-1640cell culturemedium containing kayadiol of final concentration10μmol/L for48hours.After collected by centrifugation and washed twice with PBS, the cells weretreated with100μL of dissociation buffer by severe concussion and ultrasonicfor1min in ice bath. The protein was denatured by heating to100℃for10min and centrifugated at a speed of12000r/min for10min. Then the proteinconcentration of supernatant was determined.50μg of protein was taken andseparated on SDS-PAGE gel and transferred to a nitrocellulose membrane.Incubated the membrane with5%non-fat dry milk in TBS-T for2hours at room temperature to blocked the non-specific binding and washed it with1×TBS-T for15min. Incubated the membrane with anti-Bax polyclonal antibody(1:500) and anti-caspase-3polyclonal antibody (1:700) dilution overnight atroom temperature, and washed it with1×TBS-T for3×15min. Then incubatethe membrane with secondary antibody dilution (anti-mouse1:5000) in TBS-Tfor40min and washed it with1×TBS-T for3×15min again. After incubatedwith ECL solution for1min and exposed for1¹0minnuts, the X ray filmwas developed.4The reversal effect of caspase inhibitorsHEC-1cells at the stage of logarithmic phase were inoculated in96wellplates, each well50μL (containing1×10⁴cells) and were treated for48hourswith kayadiol of final different concentration1,10and100μmol/L containingcaspase inhibitors of the final concentration20μmol/L and kayadiol of finaldifferent concentration1,10and100μmol/L not containing caspase inhibitorsin saturated humidity,37℃and5%CO2incubator. Each group was equippedwith3duplicate holes. Each hole was added5mg/mL MTT10μL before4hours to the end of cultivation. Then discarded culture supernatant carefully,added reaction stop solution150μL, and oscillated10min to dissolve thecrystallization fully. Finally the hole absorbance (OD) values was detected byEnzyme-linked immunosorbent Detector, and calculated the cell survival rate.Results:1The results of MTT assayThe compounds from the Torreya nucifer L. significantly inhibited thegrowth of cultured gynecological tumor cells in vitro. There were differentinhibitory effects on the proliferation of human gynecological tumor cellsbetween18-hydroxyferruginol, hinokiol and kayadiol, in which HEC-1,HeLa, HOC-21, HAC-2and SHIN-3. kayadiol had remarkable effect ofinhibitory proliferation on5tumor cells. After administration of kayadiol withdifferent concentrations of1,10and100μmol/L for48h, survival rate ofHEC-1cell was69.87%,39.31%and2.76%; HeLa was89.87%,41.31%and6.76%; HOC-21was73.56%,40.15%and17.27%; HAC-2was74.44%, 50.90%and11.15%; SHIN-3was69.24%,43.53%and12.49%, respectively.Administration of1-100μmol/L kayadiol might significantly inhibitproliferation of HEC-1, HeLa, HOC-21, HAC-2and SHIN-3in adose-dependent manner. There was significant difference between kayadiolgroup with the concentration of10μmol/L and control group (P<0.05),remarkable significant difference between kayadiol group with theconcentration of100μmol/L and control group (P<0.01), with the IC50of3.33μmol/L,8.00μmol/L,5.95μmol/L,7.20μmol/L and5.12μmol/L,respectively (Fig.2and Table1).2The different apoptotic rate in HEC-1cell induced by kayadiolUsing10mol/L of kayadiol treated HEC-1cells to observe the apoptosis,the results showed that cell apoptotic rate was (2.40±1.20)%,(40.16±6.84)%and (86.88±6.00)%in the solvent control group, the12h and24hgroups, respectively. Kayadio might obviously induce HEC-1cell apoptosis,and have a good time dependence (Fig.3, A and B).3Up-regulation of Caspase-3protein and Bax protein in HEC-1cellsinduced by kayadiolIn order to further investigate the effect of kayadio for Bax and caspase-3protein on HEC-1cells, using kayadio to treat HEC-1cells for48h,intracellular Bax and caspase-3protein changes were detected by Westernblotting method. The results showed that intracellular Bax and caspase-3protein expression were significantly higher after48h (Fig.4, A, B and C).4The reversal effect of caspase inhibitorsThe results showed that the survival rate of HEC-1cell which was treatedwith kayadiol of final different concentration1μmol/L,10μmol/L and100μmol/L for48h were69.87%,39.31%,2.76%. While the cell survival rateswhich were incubated with20μmol/L caspase inhibitors and kayadiol of finaldifferent concentration1,10and100μmol/L for48h increased to80.78%,36.27%and8.98%. In the concentration of1and100μmol/L of cell survivalrates increased significantly (Fig.5).Conclusion: kayadiol might significantly inhibite the proliferation of HEC-1cells, and its mechanism may be related to apoptosis. But apoptosis isa process which is induced by multiple signals, regulated by multi-gene andinvolved many factors. This experiment is just one of the pathways ofapoptosis. It is unknown that whether Kayadio is also through other pathwaysto induce apoptosis. Therefore, the mechanism of kayadio in apoptosis ofgynecologic tumor cell remain unknow. It is need for further research.