Effects and mechanism of bakuchiol-induced S-phase cell cycle arrest in human breast cancer MCF-7 cells / 中草药

Objective: To investigate the cell growth inhibitory effect and molecular mechanism of bakuchiol against human breast cancer MCF-7 cells. Methods: The growth inhibitory effect of bakuchiol on MCF-7 cells was tested by MTT assay. Flow cytometry was used to investigate the distribution of cell cycle and ROS generation. Fluorescence microscope was used to observe the change of cell nucleus. Western blotting was used to detect the expression of the protein related to cell cycle and MAPK family. The ROS scavenger and inhibitors of MAPK family were introduced to investigate the effect on the growth inhibitory rate and the levels of cell cycle related protein by bakuchiol. Results: Bakuchiol inhibited the cell growth on the MCF-7 cells in dose- and time-dependent manner, which showed stronger effect than that of 5-fluorouracil. Furthermore, bakuchiol induced S-phase arrest in MCF-7 cells via ROS generation. The production of ROS up-regulated p-p53 and p21 expression, and then decreased CDK2 and CyclinA2. The changes of bakuchiol on these proteins could be reversed by the ROS scavenger Trion, indicating that ROS was associated with bakuchiol-induced S-phase arrest. In addition, pretreatment with p38MAPK inhibitor SB203580 decreased bakuchiol-caused ROS generation, suggesting that the production of ROS was dependent on p38MAPK pathway. Conclusion: The proliferation inhibitory effect of bakuchiol on MCF-7 cells is related with S-phase cell cycle arrest, and ROS plays a role in the bakuchiol-induced S-phase arrest.

Effects of Active Constituents of Sinopodophylli Fructus on Cell Proliferation,Cell Cycle and Mitochon-drial Membrane Potential of Human Breast Cancer Cell / 中国药房

OBJECTIVE:To explore the effects and mechanism of extracts,active constituents and constituent combination of Sinopodophylli Fructus on cell proliferation of human breast cancer. METHODS:Acid phosphatase method was conducted to deter-mine the effects of 4 extracts [ethanol extract (Xc),petroleum ether extract from ethanol extract (Xp),ethyl acetate extract from ethanol extract (Xe),n-butanol extract from ethanol extract (Xz)],5 active constituents [podophyllotoxin (S1),deoxypodophyllo-toxin (S2),4-desmethyl deoxypodophyllotoxin (S3),8-isopentenyl kaempferol (S4),8,2′-diisoprenyl quercetin-3-methyl ether (S5)] and 3 active constituent combination [combination 1,S1-S2-S3-S4-S5 (2:4:1:4:32),Z1;combination 2,S2-S4 (1:1),Z2;combination 3,S3-S4(1:4),Z3] on the MDA-MB-231,MCF-7 cell proliferation;flow cytometry was adopted to detect the effects of above-mentioned samples on MDA-MB-231,MCF-7(T47D)cell cycle and mitochondrial membrane potential. RESULTS:The active constituent combination Z1 showed significant inhibitory effects on MDA-MB-231,MCF-7 cells,the half inhibitory concen-trations(IC50)were(0.27±0.2),(0.11±0.1)μg/mL;extracts Xc,Xp,Xe,active constituents S2,S4 and active constituent combi-nation Z2,Z3 showed relatively strong inhibitory effects on MDA-MB-231,MCF-7 (T47D) cell proliferation (IC50<15 μg/mL). Both extracts and active constituents can block MDA-MB-231,MCF-7 cell cycle in G2/M phase;all active constituents can block MDA-MB-231,T47D cell cycle in G0/G1 phase,and can reduce MDA-MB-231,T47D cell mitochondrial membrane potential. CONCLUSIONS:The active constituents and constituent combination of Sinopodophylli Fructus can inhibit cell proliferation of breast cancer by affecting cell cycle and mitochondrial mem-brane potential.

Anti-carcinogenic actions of glycoprotein conjugated with isoflavones from submerged-liquid culture of Agaricus blazei mycelia through reciprocal expression of Bcl-2 and Bax proteins / 동물의과학연구지

Glycoproteins isolated from fruit bodies and mycelial cultures of mushrooms exhibit anti-carcinogenic actions in human cancer cells and animal tumor cells by induction of apoptosis. Here, we report that isoflavone-conjugated glycoproteins (designate Gluvone), exhibit strong anti-carcinogenic effects on human breast cancer MCF-7 cells by induction of apoptosis. Gluvone with 9.4 kDa of molecular weight was isolated from submerged-liquid culture of Agaricus blazei mycelia (ABM) in soy flake-containing liquid medium. MCF-7 cells were incubated with various amounts of Gluvone (0~250 microM) for a period of 6 days. Gluvone exhibited anti-proliferative actions in a dose-dependent manner and 62% growth inhibition at 200 microM for 4 days relative to control. Hoechst 33258 staining analysis revealed that Gluvone induced formation of apoptotic bodies. Gluvone was associated with down-regulation of anti-apoptotic Bcl-2 protein expression as well as up-regulation of pro-apoptotic Bax protein expression. Gluvone treatment induced proteolytic activation of caspase-9 and caspase-3 through cytochrome c release from mitochondria to cytosol as well as concomitant degradation of poly (ADP-ribose) polymerase (PARP). In addition, Gluvone induced activation of caspase-8. Taken all together, these results indicate that the anti-proliferative effect of Gluvone is associated with induction of apoptotic cell death through the mitochondrial dysfunction pathway mediated by enhancement of Bax protein expression and suppression of Bcl-2 protein expression.

Inhibition of vinorelbine on invasion and metastasis of breast cancer cell and its mechanism / 中草药

The MCF-7 and MDA-MB-435 cells were treated with 1 and 10 μmol/L VRB. The cell adhesion was tested by MTS, the invasion was detected by Transwell, secretion of TGF-β was detected using ELISA, the activities of MMP-2 and MMP-9 were detected by zymography, the expression of proteins, including E-cadherin (E-CAD), N-cadherin (N-CAD), MMP-2, and MMP-9, p-JNK and, p-Akt was evaluated by Western blotting, RT-PCR was used to detect E-CAD, N-CAD, MMP-2, and MMP-9 genes, and dual-luciferase reporter assay was used to validate the activities of AP-1 and NF-κB. Results After being treated with 1 and 10 μmol/L VRB, for MCF-7 and MDA-MB-435, the adhesion ability was decreased by 34.8% and 66.8%, 42.6% and 72.3%; The metastatic ability was decreased by 44.4% and 72.2%, 47.7% and 86.4%; The secretion of TGF-β was reduced by 28.2% and 52.1%, 39.0% and 55.1%, significantly; The mRNA expression levels of E-CAD were increased by 86.5% and 181.6%, 116.6% and 160.7%; while the levels of N-CAD were decreased by 33.7% and 74.1%, 57.6% and 76.9%; The gene expression of MMP-2 was decreased by 71.6% and 88.4%, 57.4% and 69.4%; The gene expression of MMP-9 was decreased by 15.0% and 84.0%, 22.1% and 73.0%, respectively. The protein expression of E-CAD was up-regulated while the protein expression of N-CAD, MMP-2, MMP-9, p-JNK, and p-Akt were down-regulated significantly; And dual-luciferase reporter assay validated VRB could inhibit the transcriptional activity of AP-1 and NF-κB by 27.7% and 68.2%, 34.8% and 71.4%, 18.4% and 44.8%, 24.4% and 51.9%, respectively. Conclusion VRB could inhibit the metastasis of breast cancer MCF-7 and MDA-MB-435 cells via down-regulation of inhibition and blocking of signaling pathway correlated with metastasis and epithelial-mesenchymal transition.