Signal Transduction Pathways Of Apoptosis Induced By Swatnsonine In Caprine Luteal Cells

Swainsonine (1,2,8-trihyroxyindolizidine, SW), a natural toxin, is an indolizidinealkaloid isolated from locoweed a number of poisonous plants including Astragalus andOxytropis species. Ingestion of SW-containing plants not only cause livestock death, but alsoresult in infertility or abortion in the livestock, which causes tremendous loss annually to thelivestock industry. Up to now, the molecular mechanisms for SW causes female abortion isstill unknown. Recent years, many researchers have demonstrated that some toxicologicalcompounds can induce luteal cell apopotsis and cause abnormal luteal function throughinhibition of progesterone production, resulting in reproductive failure. Therefore, in thisstudy, we firstly established a transfected steroidogenic caprine luteal cell line, which wasused to investigate the effects of SW on progesterone secretion and cell viability and themechanisms involved in these processes in vitro. Finally, the exact molecular pathways ofSW-induced apoptosis were studied scientificly. The results were as follows:1. Caprine corpora lutea (CL) judged to be in early (6to8weeks) stages of pregnancy werecollected. Luteal cells were isolated from CL by2mg/mL collagenase digestion. Luteal cellsobtained was collected by centrifugation, cultured in DMEM/DF12supplemented with10%fetal calf serum at37oC and5%CO2. The cells became adherent after12h culture. Twoshapes cells were found, spindle and polygonal. Cytochemistry staining showed that this2shapes cells were positive for3β-hydroxysteroid dehydrogenase (3β-HSD) activity, a markerfor steroidogenic cells, with95%of positive rate. Primary luteal cells were transfected with aplasmid pCI-neo-hTERT containing the human telomerase reverse transcriptase (hTERT)gene by Lipofectamin2000. Clones were selected after450μg/mL G418resistance for14d,and positive clones were amplified. After purifying by magnetic cell separation kit, thetransduced luteal cells at passage30and50grew as confluent monolayers with the typicalspindle and spherical shaped morphology, which were similar to primary luteal cells atpassage. Cytochemistry staining showed that the transfected luteal cells at passage30and50were positive for3β-HSD activity. Results from RT-PCR and TRAP-ELISA assay confirmed that transduced luteal cells steadily expressed hTERT gene and exhibited higher telomeraseactivity at passage30and50. Up to now, the cells have been cultured for60passages in vitro.Taken together, our results demonstrate that over-expression of hTERT in caprine luteal cellscan reactivate telomerase and immortalize luteal cells.2. RT-PCR showed that the transfeceted luteal cells at passage30and50expressed genesencoding key proteins, enzymes and receptors inherent to normal luteal cells, such assteroidogenic acute regulatory protein (StAR), cytochrome P450cholesterol side-chaincleavage enzyme (P450scc),3β-HSD and luteinizing hormone receptor (LH-R). In addition,radioimmunoassay (RIA) showed that the transfeceted luteal cells produced detectablequantities of progesterone upon8-bromo-cAMP (8-Br-cAMP) or22(R)-hydroxycholesterol(22R-HC) stimulation. The growth curve showed that the transfected luteal cells grow morerapidly than primary luteal cells (Population Doubling Time (PDT):23.5:24.2h). Karyotypeanalysis showed that the transfected luteal cells have normal chromosome complement with amodal chromosome number of60. Furthermore, Soft agar assay and the xenograft in nudemice showed that the transfected luteal cells have no neoplastic transformation in vitro and invivo. Thes results demonstrated that immortalized luteal cells by hTERT retain their originalcharacteristics without neoplastic transformation, and may provide a useful model for studiesof luteal cell functions.3. Transfected and primary luteal cells were used to investigate the effects of SW onprogesterone secretion and cell viability. RIA showed that higher concentrations of SW (3.2and4.8μg/mL) treatments significantly inhibited progesterone production in the presence orabsence of22R-HC and pregnenolone in both transfected and primary luteal cells at24and48h when compared to the control group. However, SW at lower concentrations (0.4,0.8and1.6μg/mL) significantly stimulated the basal progesterone production and enhanced22R-HC-stimulated progesterone production when compared to the control group, while0.8and1.6μg/mL of SW significantly promoted pregnenolone-stimulated progesteroneproduction when compared to the control group in both transfected luteal cells and primaryluteal cells. MTT assay showed SW concentrations were lower than1.6μg/mL within24h or0.8μg/mL within48h. However, with the increase of treatment time and SW concentrations,cell viability significantly decreased when compared to the cells without SW treatment.4. Results from QRT-PCR and Weseter blot assay showed that StAR did not showsignificant changes at both mRNA and protein levels in different concentrations of SW-treatedcells compared to control cells. However, the expression of P450scc significantly increased atboth mRNA and protein levels in the luteal cells treated with0.4,0.8and1.6μg/mL of SWfor24h, compared to the control. The expression of3β-HSD significantly increased at both mRNA and protein levels in the luteal cells treated with0.8and1.6μg/mL of SW for24h. Inaddition, flow cytometry showed that after24h treatment, the percentage of cells in G0/G1phase increased from51%in the control group cells to63%and68%in the cells treated with3.2and4.8μg/mL of SW, respectively. After48h treatment, SW at0.8,1.6,3.2and4.8μg/mL significantly altered the cell cycle distribution, compared to the control group. Thepercentage of cells at G0/G1phase increased from52%to81%. Results from Western blotassay further confirmed that higher concentrations of SW induced luteal cell cycle arrest inG0/G1through down-regulating the expression of Cyclin E, Cyclin D1, CDK2, andup-regulating the p21protein levels. Annexin V-FITC/PI staining assay showed that over1.6μg/ml of SW induced cell apoptosis after48h treatment, and over3.2μg/ml of SW inducedcell apoptosis after24h treatment. DNA fragmentation assay showed that the cells treatedwith4.8μg/mL SW for48h showed a typical DNA ladder pattern.5. Caspase activity analysis showed that SW treatment increased the activities of Caspase-9and Caspase-3without affecting that of Caspase-8. After Caspase-3activation, the full-length(116kDa) poly ADP ribose polymerase (PARP) were cleaved to active form of85kDa, ahallmark of apoptosis. z-VAD-fmk (pan caspase inhibitor), z-LEHD-fmk(caspase-9specificinhibitor), or z-DEVD-fmk (caspase-3specific inhibitor) significantly inhibited SW-inducedapoptosis, whereas z-IETD-fmk (caspase-8inhibitor)did not, suggested SW-induced apoptosisdependent on Caspase-9and Caspase-3activation. However, the expression of Fas, Fas ligand(FasL) or caspase-8activity did not appear significant changes in the process of SW-inducedapoptosis. Both QRT-PCR and Western blot assay showed that SW treatment up-regulatedBax, down-regulated Bcl-2expression, promoted Bax translocation to mitochondria, activatedmitochondria mediated apoptotic pathway, which in turn caused the release of cytochrome c,the activation of caspase-9and caspase-3. In addition, SW treatment did not affect theexpression levels of Smac and AIF in the mitochondrial fractions.Taken together, the present study we established and evaluated a stable steroidogeniccaprine luteal cell line through transfection of a plasmid containing the hTERT gene. Theimmortalized luteal cells still retained their original characteristics, including steroidbiosynthesis capability, and the expression of key steroidogenic genes that are modulated bydifferent physiologic inducers, and will provide an important tool for the study of corpusluteum function. In addition, this study also illustrated that lower concentrations of SWinduced progesterone production through up-regulation of P450scc and3β-HSD withoutaffecting cell viability, whereas higher concentrations of SW induced cell cycle arrest andapoptosis throuth activation of mitochondrial pathway. The results may be a potentiallyvaluable and reliable cell model to be used to study function and regulation of normal caprine luteal cells. Furthermore, it could be used as an in vitro model to assess the effects of manyphysiological, biological, pharmacological and toxicological events and compounds on CLfunction. The results may also provide theoretical basis for prevention and control livestockpoisoning caused by toxic plants that serious damage to the grassland of west China.