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OBJECTIVE To investigate the effects of polyphyllin Ⅵ（PPⅥ） on the proliferation and apoptosis of glioma cells and potential mechanism. METHODS Using human glioma LN229 cells as objects， MTT assay was used to detect the survival rate after treated with different concentrations of PPⅥ [0 （control group）， 1， 2， 4， 8， 16， 32， 64 μmol/L] for different time （24， 48， 72 h）. The clone formation experiments were adopted to detect the number of cell clones and clone formation rate after being treated with different concentrations of PPⅥ [0 （control group）， 2， 4， 8 μmol/L] for 14 days. The flow cytometry and Western blot assay were used to detect the apoptotic rate of cells， the expressions of apoptosis-related protein [B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， cleaved caspase-3]， and the expressions of related proteins of Fas/Fas ligand （FasL） death receptor pathway and protein kinase B （Akt）/glycogen synthesis kinase-3β （GSK-3β） pathway after being treated with different concentrations of PPⅥ [0（control group）， 4， 8 μmol/L] for 24 h. RESULTS Compared with the control group， the survival rate of cells， the number of clones and clone formation rate， the protein expression of Bcl-2， and the phosphorylation levels of Akt and GSK-3β protein were decreased significantly in different concentration groups of PPⅥ （P＜0.05 or P＜0.01）. The apoptotic rate， the protein expressions of Bax， cleaved caspase-3， Fas， FasL and cleaved caspase-8 were increased significantly （P＜0.05 or P＜ 0.01）. CONCLUSIONS PPⅥ can inhibit the proliferation and induce the apoptosis of human glioma LN229 cells， which may be related to the activation of the Fas/FasL death receptor pathway and the inhibition of the Akt/GSK-3β pathway.
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Background 2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used as a broad-leaved herbicide and plant growth regulator. Related studies have shown that 2,4-D has neurotoxicity, ability to disrupt endocrine function, genotoxicity, carcinogenicity, and reproductive toxicity. Objective This experiment is conducted to investigate the effect of 2,4-D exposure on reproductive system of female rats, and to preliminarily explore the potential ameliorative effect of Lycium barbarum polysaccharide (LBP) and its possible mechanism. Methods Twenty-four SPF female SD rats with six rats in each group were randomly divided into a blank control group (deionized water 1 mL·d−1), an exposure group (75 mg·kg−1 2,4-D), an LBP control group (50 mg·kg−1 LBP), and an LBP intervention group (75 mg·kg−1 2,4-D + 50 mg·kg−1 LBP). The rats were given intragastric administration once a day for 28 consecutive days. Body weight was measured every two days. After exposure, ovary and uterus were weighed and organ coefficients were calculated; the pathological changes of ovary and uterus were detected by hematoxylin-eosin staining (HE); the level of estradiol (E2) in serum was detected by ELISA; the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in serum were measured by corresponding kits; the apoptosis of ovarian and uterine cells was detected by TUNEL fluorescence staining; and the protein expression levels of Fas, FasL, FADD, Pro-Caspase-8, Cleaved-Caspase-8, Pro-Caspase-3, and Cleaved-Caspase-3 in ovarian tissues were detected by Western blotting. Results Compared with the blank control group, the ovarian structure of the exposure group was abnormal, the number of follicles at different developmental stages decreased, morphological changes were observed, and the number of atretic follicles increased; the endometrium was incomplete, with different degrees of nuclear pseudostratification and decreased number of glands in lamina propria. Compared with the exposure group, the ovarian structure of the LBP intervention group was complete, and the follicles at different developmental stages increased in amount, remained intact, and were arranged closely; the uterine structure was relatively intact, showing decreased endometrial loss and nuclear pseudostratification. There were significant differences in the levels of SOD, GSH-Px, E2, and MDA among the four groups (F=86.1, 26.2, 43.3, and 22.3, all P<0.01). Compared with the blank control group, the levels of serum SOD, GSH-Px, and E2 decreased in the exposure group (P<0.01), while the concentration of MDA increased (P<0.01). Compared with the exposure group, the levels of serum SOD, GSH-Px, and E2 in the LBP intervention group increased (P<0.01), and the concentration of MDA decreased (P<0.01). There were significant differences in the apoptosis rates of ovarian and uterine cells among the four groups (F=64.8, 55.5, both P<0.01). Compared with the blank control group, the apoptosis rates of ovarian and uterine cells increased in the exposure group (P<0.01). Compared with the exposure group, the apoptosis rates of ovarian and uterine cells decreased in the LBP intervention group (P<0.01). There were significant differences in the expression levels of death receptor pathway-related proteins in ovarian tissues among the four groups (all P<0.05). Compared with the blank control group, the expression levels of Fas, FasL, FADD, Pro-Caspase-8, Cleaved-Caspase-8, Pro-caspase-3, and Cleaved-Caspase-3 were increased in the exposure group (P<0.05 or 0.01). Compared with the exposure group, the expression levels of above proteins were decreased in the LBP intervention group (P<0.05 or 0.01). Conclusion The study findings reveal that 2,4-D can induce oxidative stress and further mediate Fas-FasL pathway to induce apoptosis, resulting in reproductive system damage in female rats. LBP can reduce the oxidative stress level, down-regulate the expression of Fas-FasL pathway-related proteins, and reduce the apoptosis of germ cells, therefore protecting reproductive system of female rats.
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Objective: To investigate the effects of xanthotoxin from Apiaceae medicinal plants on cell proliferation and apoptosis, and explore its mechanism of action against human gastric carcinoma SGC-7901 cells in vitro. Methods: SGC-7901, HepG-2, MCF-7, and A549 cells were treated with different concentrations of xanthotoxin (10, 20, 60, 80, 100, 120, 140, and 160 µg/mL) for 48 h, and the cell viability (IC50) was determined by MTT assay; Xanthotoxin-induced apoptosis in cells was observed by using Hoechst 33258 Staining Kit and Annexin V-FITC Apoptosis Detection Kit; Flow cytometry was used to detect apoptosis related proteins of Fas/FasL, Bid, and DR5/TRAIL proteins in human gastric carcinoma SGC-7901 cells after being treated by xanthotoxin; The influence of xanthotoxin on Caspase-8 protein expression in the cells was determined by Flouormetric Assay Kit. Results: Xanthotoxin obviously inhibited SGC-7901, HepG-2, MCF-7, and A549 cells proliferation, and its inhibition was in a concentration-dependent manner; flow cytometry results showed that in a certain concentration range, xanthotoxin can increase the expression levels of Fas/FasL and DR5/TRAIL proteins in a concentration-dependence manner. The content of Bid protein in cells was increased, and it showed concentration-dependence. Conclusion: Xanthotoxin may induce SGC-7901 cells apoptosis in a certain concentration range through the Fas/FasL protein mediated death receptor pathway, or by DR5/TRAIL mediated death receptor pathway, and increase the expression level of death receptor protein, activation Caspase-8, activating downstream effect factor, inducing cell apoptosis, or activate Caspase-8 cutting activate protein Bid, and then enter the mitochondrial pathway, induction of apoptosis.
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Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.