| Object：To investigate the molecular mechanism of20(S)-protopanaxadiol (PPD)inducing human breast cancer MCF-7cells apoptosis based on PI3K/AKT/mTORsignaling pathway.Methods：The effect of PPD on human breast cancer MCF-7cells proliferation weredetected by MTT assay, and calculated IC50value of24h. The morphologicalchanges of MCF-7cells were detected by fluorescence microscope after AO/EB andDAPI staining. The mitochondrial transmembrane potential, the apoptosis rate andcell cycle were detected by fluorescence microscope; Caspase-3and Caspase-9activity were determined by spectrophotometry; After adding Caspase family’sextensive inhibitor z-VAD-fmk, the apoptosis rate was detected; Caspase-3, Caspase-9, PARP, Bcl-2, Bax, p53, p27kip1, C-myc, CDK4and Cyclin D1protein expressionwere determined by Western Blot; Bcl-2and Bax gene level were detected by QPCR.The effect of PPD on PI3K/AKT/mTOR signaling pathway associated proteinAKT, P-AKT (Thr308), P-AKT (Ser473), PTEN, P-PTEN (Ser380), GSK-3β,P-GSK-3β (Ser9), mTOR, P-mTOR (Ser2448) and downstream target proteinsP-FoxO1(Ser256), P-MDM2(Ser166) P-NF-κB p65(Ser536) protein expressionwere detected by Western Blot. After transient transfection of mTOR plasmid andmTOR siRNA in vitro, MCF-7cells were treated with PPD for24h, then the survivaland apoptosis rate were detected, and the mTOR phosphorylation level and Bcl-2andBax protein expression were detected by Western Blot. Establish the transplantation tumor model of MCF-7cells in nude mice. Observethe effect of PPD on tumor inhibition rate, organ index, tumor tissue HE staining,TUNEL staining; and P-mTOR, P-AKT, Bax, Bcl-2expression were detected byimmunohistochemical assay.Results：1. PPD induced MCF-7cells apoptosis through mitochondrial pathwayPPD inhibited MCF-7cells proliferation significantly in a dose-dependent manner.IC50value of24h was33.33μM; MCF-7cells were treated with PPD (0,15,30and60μM) after24h, observed under optical microscope, the number of MCF-7cellswas reduced significantly, the gradual emergence of suspension cells and refractionwas getting worse; Fluorescence inverse microscope observation demonstrated that,with increasing concentrations of PPD, a growing number of apoptotic cells werestained EB issued orange or orange-red fluorescence after AO/EB staining; Thecondensation of nuclear chromatin, blue fluorescence enhancement andkaryopyknosis were appeared after DAPI staining; After Annexin V/PI staining,with increasing concentrations of PPD, the apoptosis rate increases and ΔΨm reducesfrom (83.65±8.21)%to (48.37±3.12)%significantly in a dose-dependent detected byflow cytometry; PPD can induce activity of Caspase-3and Caspase-9on MCF-7cellsincreased with increasing concentration of PPD significantly in a dose-dependentmanner; After adding the Caspase family’s extensive inhibitor z-VAD-fmk,PPD-induced apoptosis rate was significantly decreased, PPD (30μM)-inducedapoptosis rate was (43.72±2.96)%, after adding inhibitor z-VAD-fmk, the apoptosisrate was (19.54±2.35)%, there was a significant difference compared with PPD(P<0.05); With increasing concentration of PPD, expression of Procaspase-3andProcaspase-9protein decreased, PARP protein hydrolyzate specificity increased;Western Blot and QPCR detection demonstrated that, with increasing concentrationsof PPD, protein and gene expression of Bax was increased, protein and gene expression of Bcl-2was decreased and Bax/Bcl-2ratio was increased significantly.2. PPD induced MCF-7cells G0/G1phase arrestPercentage of cells in G0/G1phase with the PPD (0,15,30and60μM) were(53.89±8.55)%,(56.62±7.62)%,(61.31±8.12)%,(74.61±4.67)%, in a dose-dependentmanner; and the expression of p53protein was increased, expression of p27kip1,C-myc, CDK4and Cyclin D1was decreased.3. PPD induced PI3K/AKT/mTOR signaling pathway inhibition on MCF-7cellsAfter treated with PPD (0,15,30and60μM) on MCF-7cells for24h, WesternBlot detection demonstrated that, with the increase of the concentration of PPD,P-PTEN protein expression was increased, P-AKT (Thr308), P-AKT (Ser473),P-GSK-3β (Ser9), P-mTOR (Ser2448), P-FoxO1(Ser256), P-MDM2(Ser166) andP-NF-κB p65(Ser536) protein expression was decreased, AKT, PTEN, GSK-3β andmTOR protein expression showed no significant change.4. The effect of mTOR plasmid on PPD-induced apoptosis in MCF-7cellsCell viability rate of PPD (30μM) was (63.97±3.81)%, cell viability rate of PPDcombined with mTOR plasmid was (75.13±2.67)%, there was a significant differencecompared with PPD (P<0.05). Apoptosis rate of PPD (30μM) was (37.30±3.76)%,while the apoptotic rate of PPD combined with mTOR plasmid was (19.73±2.99)%,there was a significant difference compared with PPD (P<0.05). In addition, aftertreated with PPD (30μM) and mTOR plasmid, phosphorylation level of mTOR, Bcl-2protein expression were higher than PPD, and Bax protein levels lower than PPD.5. The effect of mTOR siRNA on PPD-induced apoptosis in MCF-7cellsCell viability rate of PPD (30μM) was (50.17±1.90)%, cell viability of PPDcombined with mTOR siRNA was (34.13±2.05)%, there was a significant differencecompared with PPD (P<0.05). Apoptosis rate of PPD (30μM) was (27.20±1.90)%,apoptosis rate of PPD combined with mTOR siRNA was (43.07±3.15)%, and therewas a significant difference compared with PPD (P<0.05). In addition, after treated with PPD (30μM) and mTOR siRNA, phosphorylation level of mTOR, Bcl-2proteinexpression was lower than PPD, and Bax protein expression was higher than PPD.6. Anti-tumor effect of PPD on MCF-7cells xenografts nude mice and its mechanismPPD (50,100mg·kg-1) inhibited transplantation tumor of xenografts nude micegrowth in a dose-dependent manner; increased the heart, liver, lung, kidney index,while decreased spleen index. Tumor tissue HE staining demonstrated that, aftertreated PPD (50mg·kg-1), tumor cells were arranged in tight, cytoplasm stained moredeeply, nucleus was clear, a few of hemorrhage and necrosis cells appeared; whiletreated PPD (100mg·kg-1), tumor cells were arranged in loose, cytoplasm stained lessdeeply, nucleus was clear, a large number of hemorrhage and necrosis cells appeared.Tumor tissue TUNEL staining demonstrated that PPD (50,100mg·kg-1) induced tumortissue apoptosis (P<0.05). The result of immunohistochemistry analysis demonstratedthat PPD (50,100mg·kg-1) decreased P-mTOR, P-AKT and Bcl-2expression levels,increased Bax expression level.Conclusions：1. PPD significantly inhibits MCF-7cells proliferation and induced apoptosis in adose-dependent manner.2. PPD decreases the level of Bcl-2expression, increases the level of Baxexpression, regulates Bax/Bcl-2ratio to decrease mitochondrial membrane potential,activates Caspase protein family, prompts PARP cleaved, induces MCF-7cellsapoptosis through mitochondrial pathway at protein and gene level.3. PPD induces G0/G1phase of MCF-7cells by upregulating the expression ofp53and downregulating the expression of p27kip1, C-myc, Cyclin D1and CDK4.4. PPD induces MCF-7cells apoptosis by inhibiting the PI3K/AKT/mTOR signaltransduction pathway.5. PPD shows anti-tumor effect on transplantation tumor of breast carcinoma cellline MCF-7in nude mice and its mechanism probably concerned with inhibition of PI3K/AKT/mTOR signaling pathway. |
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