ABSTRACT
The purpose of this research is to identify the chemical constituents of sea buckthorn leaves extract (SBLE) and explore its hypoglycemic biological activity. SBLE was prepared by hot reflux extraction with 65% ethanol, and its chemical composition was analyzed by ultra-high-performance liquid chromatography-photodiode array-mass spectrometry/mass spectrometry (UHPLC-PDA-MS/MS) system. The animal experiments were compliant with ethical principles for animal use and had been approved by the Animal Experiment Ethics Committee of Jinan University. Mice were injected with streptozocin (STZ) to establish a hyperglycemic animal model, and SBLE (1.5 g·kg-1) was administered by gavage for 5 weeks. The fasting blood glucose (FBG) and oral glucose tolerance were detected. Normal mice were given SBLE (1.5 g·kg-1) by intragastric administration for 10 days, and blood was collected from the tail vein to detect the changes in blood glucose within 120 min after sucrose or starch loading. The mucous membrane of the small intestine of mice was taken to detect the activity of α-glucosidase (AG), and the activity of yeast-derived AG incubated with SBLE was evaluated. The glucose uptake by Caco-2 cells treated with SBLE was detected by fluorescence microscopy and cytometry, and the gene expression of sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) in Caco-2 cells were detected by real-time quantitative PCR (qPCR). A total of 18 compounds were identified, mainly including tannins and flavonoids. SBLE reduced FBG and increased oral glucose tolerance in STZ hyperglycemic mice. SBLE effectively inhibited the increase of blood glucose caused by starch intake in normal mice. SBLE exerted good inhibitory activity on yeast-derived AG (IC50 = 16.94 μg·mL-1) and small intestinal mucosa AG with an inhibition rate of 15.48%. SBLE (25-100 μg·mL-1) dose-dependently inhibited glucose uptake by Caco-2 cells, and SBLE significantly reduced the mRNA level of SGLT1 without changing the expression of GLUT2. In conclusion, the UHPLC characteristic fingerprint of SBLE is established with 18 chemical components identified by mass spectrometry, and SBLE exerts hypoglycemic effect by inhibiting the activity of AG and the absorption of glucose by intestinal epithelial cells.
ABSTRACT
OBJECTIVE@#To investigate the mechanism of Paris forrestii (Takht.) H. Li (PCT3)-suppressing the proliferation of HL-60, K562, KG-1 and HT-93 cells.@*METHODS@#cute myeloid leukemia cell lines such as HL-60, K562, KG-1 and HT-93 were treated with Paris forrestii (Takht.) H. Li (PCT3) for 24, 48, and 72 h, and MTT assay was employed to determine the cells proliferation. Meanwhile, the apoptosis of K562, HL-60, KG-1 and HT-93 cells were detected by flow cytometry after PCT3 (Control, 4 μg/ml, 8 μg/ml) treated for 24 h and the Western blot was performed to detect the expression of PARP,Caspase-3, MCL-1, BAX, BCL-2, P53, and P27. GAPDH was used as an internal loading control.@*RESULTS@#MTT assay showed that Paris forrestii (Takht.) H. Li (PCT3) significantly inhibited the proliferation of HL-60, K562, KG-1 and HT-93 cells in concentration and time-dependent manners. Compared with the control group, the leukemia cell viabilities were significantly suppressed (r =0.9436; r =0.8623; r =0.9922; r =0.8918). Paris forrestii (Takht.) H. Li (PCT3) induced apoptosis of leukemia cells in a concentration dependent manner, compared with the control group (P＜0.05 or P＜0.01). Western blot revealed that PARP, a major enzyme in DNA damage repair, and Caspase-3 another one of the major executive apoptotic enzymes were cleaved in cell lines examined, and this cleavage was concentration dependent. Anti-apoptotic proteins such as MCL-1 and BCL-2 were down regulated by Paris forrestii (Takht.) H. Li (PCT3), and Pro-apoptotic protein BAX was upregulated. And the protein of tumor suppressor gene P53 and its downstream signaling protein P27 increased.@*CONCLUSION@#Paris forrestii (Takht.) H. Li (PCT3) can inhibit the proliferation of leukemia cells by activating endogenous apoptosis pathway, and provide a potential new drug selection for clinical treatment of AML leukemia.
Subject(s)
Humans , Apoptosis , Caspase 3 , Cell Line, Tumor , Cell Proliferation , Leukemia, Myeloid, Acute , Melanthiaceae , Proto-Oncogene Proteins c-bcl-2ABSTRACT
Objective Mifepristone (MIF) can inhibit triple-negative breast cancer cell (TNBC) survival at high concentra-tions. The purpose of the present study is to study the effect of mifepristone derivatives on triple-negative breast cancer cell (TNBC) survival at low concentrations. Methods SUM149PT and HCC1937 triple negative breast cancer cells were used in the study; the experiment was set in four groups: DMSO group,MIF group(10 μmol/L MIF),5 μmol/L FZU-00,033 group and 10 μmol/L FZU-00,033 group. They were treated with 24,48,72 h,and cell viability was measured by SRB. KLF5 overexpression HCC1937 cell line was used in the study; the experiment was set in four groups: PCDH-DMSO group(PCDH vector,DMSO),PCDH-FZU-00,033 group (PCDH vector,10 μmol/L FZU-00,033),KLF5-DMSO group(overexpress KLF5,DMSO),KLF5-FZU-00,033 group(overexpress KLF5,10 μmol/L FZU-00,033). Cell apoptosis was investigated by detecting PARP cleavage using Western blot. In order to investi-gate how FZU-00,033 reduced cell viability,we detected KLF5 protein expression after drug treatment. On the basic of the original PC-DH-DMSO group,PCDH-FZU-00,033 group,KLF5-DMSO group and KLF5-FZU-00,033 group,5 μmol/L PCDH-FZU-00,033 group (PCDH vector,5 μmol/L FZU-00,033),5 μmol/L KLF5-FZU-00,033 group(overexpress KLF5,5 μmol/L FZU-00,033). Western blot was used to detect the effect of FZU-00,033 in KLF5 overexpression cell line. Results Compared with DMSO group,5 μmol/L FZU-00,033 group,10 μmol/L FZU-00,033 group and MIF group decreasd TNBC cell viability more efficiently at 24,48,72h (P<0.01); the cell survival rate of DMSO group,5 μmol/L FZU-00,033 group,10 μmol/L FZU-00,033 group and MIF group was [(100±4)%,(17±2)%,(5±1)%,(58±1)%] respectively in SUM149PT cell line and was [(100±7)%,(39±1)%,(30±1)%,(62±1)%] respectively in HCC1937 cell line. Compared with MIF group,5 μmol/L FZU-00,033 group and 10 μmol/L FZU-00,033 group decreasd TNBC cell viability more efficiently at 24,48,72 h (P<0.01). Compared with DMSO group,5 μmol/L FZU-00,033 group,10 μmol/L FZU-00,033 group and MIF group suppressed KLF5 expression more potently and increased cell apoptosis. Com-pared with 10 μmol/L MIF group,5 μmol/L FZU-00,033 group and 10 μmol/L FZU-00,033 group significantly increased apoptosis. Compared with PCDH-DMSO group,10 μmol/L PCDH-FZU-00,033 group decreasd TNBC cell viability more efficiently at 48,72 h and cell survial rate was [(100±6)% vs (39±2)%,P<0.05] and [(100±3)% vs (21±1)%,P<0.05] respectively. Compared with KLF5-DMSO group,10 μmol/L KLF5-FZU-00,033 group decreasd TNBC cell viability more efficiently at 48,72h and cell survial rate was [(100±1)% vs (47±1)%,P<0.05] and [(100±1)% vs (27±1)%,P<0.05] respectively; Meanwhile,Compared with 10 μmol/L PCDH-FZU-00,033 group,10 μmol/L KLF5-FZU-00,033 group increased TNBC cell viability more efficiently at 48,72 h (P<0.05). Compared with PCDH-DMSO group,5 μmol/L PCDH-FZU-00,033 group and 10 μmol/L PCDH-FZU-00,033 group in-creased cell apoptosis; Compared with KLF5-DMSO group,5 μmol/L KLF5-FZU-00,033 group and 10 μmol/L KLF5-FZU-00,033 group increased cell apoptosis. Conclusion Novel mifepristone derivative FZU-00,033 suppressed TNBC cell viability partially through suppressing KLF5 expression.
ABSTRACT
Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accu-mulation of immature myeloid progenitor cells in the bone marrow,compromising of normal hematopoi-esis and ultimately resulting in bone marrow failure. Chemotherapy is the mainstay treatment for all AML patients,however,drug resistance and clinical relapse limits its efficacy.The 5-year survival rate of AML patients is only 26.6%.Survival rates are even lower among patients ages 65 to 74 years (5.3%)and 75 years or older(1.6%).Therefore,exploring novel therapeutic agents is urgent for improving the outcome of patients with AML. Saponins are amphipathic glycosides found in traditional Chinese medicines. In the present study, we isolated a panel of saponins from Paris forrestii (Takht.) H. Li, a unique plant found in Tibet and Yunnan provinces, China. By examining their activities in suppressing acute myeloid leukemia cell proliferation, total saponins from Paris forrestii (TSPf) displayed more potent activity than individual ones.TSPf induced more than 40% AML cell apoptosis within 24 h and decreased the viability of all leukemia cell lines. TSPf-induced apoptosis was confirmed by both Annexin V staining and caspase-3 activation.TSPf downregulated pro-survival proteins Mcl-1,Bcl-xL and Bcl-2,but upreg-ulated the expression of tumor suppressor proteins p53,p27,Bax and Beclin 1.The AKT/mTOR signaling pathway is frequently over activated in various AML cells,and TSPf was found to suppress the activa-tion of both AKT and mTOR,but had no effects on their total protein expression.This was further con-firmed by the inactivation of 4EBP-1 and p70S6K,two typical downstream signal molecules in the AKT/mTOR pathway. More specifically, TSPf-inactivated AKT/mTOR signaling was found to be associated with downregulated RNF6, a recently identified oncogene in AML. RNF6 activated AKT/mTOR, and consistently, knockdown of RNF6 led to inactivation of the AKT/mTOR pathway. Furthermore, TSPf suppressed the growth of AML xenografts in nude mice models. Oral administration of 100 mg·kg-1 body weight almost fully suppressed tumor growth within 14 d, without gross toxicity. This study thus demonstrated that TSPf displays potent anti-AML activity by suppressing the RNF6/AKT/mTOR pathway. Given its low toxicity,TSPf could be developed for the treatment of AML.
ABSTRACT
AIM@#To study the chemical constituents and bioactivity of the seeds of Crataegus pinnatifida.@*METHODS@#The chemical constituents were isolated and purified by macroporous adsorptive resin D101, silica gel, and ODS column chromatography, and preparative HPLC. Their structures were elucidated on the basis of spectroscopic methods. In addition, the cytotoxic activities of compounds 1-4 were investigated on OPM2 and RPMI-8226 cells.@*RESULTS@#Four compounds were obtained and their structures were identified as (7S, 8S)-4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3, 5-dimethoxybenzaldehyde (1), (+)-balanophonin (2), erythro-guaiacylglycerol-β-coniferyl aldehyde ether (3), buddlenol A (4).@*CONCLUSION@#Compound 1 is a novel norlignan, while compounds 1-4 exhibited marginal inhibition on the proliferation of OPM2 and RPMI-8226 cells.