[Expression of Runt-related Transcription Factor 3 in Human Colon Cancer Cell Line HCT-116 Resistant to 5-Fluorouracil and the Mechanism of Drug Resistance].

Objective To establish a human colon cancer cell line HCT-116/5-FU resistant to 5-fluorouracil(5-FU)and explore the relationship between runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Methods The human colon cancer cell line HCT-116/5-FU with resistance to 5-FU was established by low concentration gradient increment combined with high-dose intermittent shock.CCK-8 method was used to determine the half maximal inhibitory concentration(IC50)of 5-FU on the parent line HCT-116 and drug-resistant line HCT-116/5-FU.The cell growth curve was established for the calculation of population doubling time(TD).The mRNA levels and protein levels of RUNX3,P-glycoprotein(P-gp),multidrug resistance-associated protein 1(MRP1),and lung resistance-related protein(LRP)in HCT-116 and HCT-116/5-FU cells were determined by qRT-PCR and Western blotting,respectively.The RUNX3 expression in HCT-116 cells was knocked down by siRNA technique,and the cells were divided into RUNX3 knockdown groups(si-RUNX3-1 group and si-RUNX3-2 group)and negative control group(si-NC group).The knockdown efficiency was verified by qRT-PCR at the mRNA level and Western blotting at the protein level.The IC50 in si-RUNX3 groups and si-NC group was determined with CCK-8 method,and the expression of P-gp,MRP1,and LRP in the two groups was detected by Western blotting.Results A stable human colon cancer drug-resistant cell line HCT-116/5-FU was successfully constructed.HCT-116/5-FU showed the TD 1.38 times as long as that of HCT-116(P=0.002)and changed morphology.The mRNA level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116 cells(P=0.048),and those of P-gp(P=0.008),MRP1(P=0.001),and LRP(P=0.001)showed the opposite trend.The protein level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116(P<0.001),and those of P-gp,MRP1,and LRP presented the opposite trend(all P<0.001).The HCT-116 cell model with low expression of RUNX3 was successfully established.The mRNA level of RUNX3 had no significant difference between si-RUNX3-1 group and si-NC group(P=0.064),while the level in si-RUNX3-2 group was significantly lower than that in si-NC group(P=0.034).The protein levels of RUNX3 in si-RUNX3-1 group and si-RUNX3-2 group were lower than that in si-NC group(both P<0.001).The results demonstrated higher knocking efficiency in si-RUNX3-2 group,which was thus selected to complete the follow-up test.The IC50 of si-RUNX3 group was significantly higher than that of si-NC group(P<0.001),which indicated that the down-regulated expression of RUNX3 could reduce the sensitivity of HCT-116 cells to 5-FU.The relative protein levels of P-gp,MRP1,and LRP in si-RUNX3 group were significantly higher than those in si-NC group(all P<0.001).Conclusion The down-regulation of RUNX3 expression can reduce the sensitivity of HCT-116 cells to 5-FU,which is considered to be related to the up-regulated expression of P-gp,MRP1,and LRP.

Ginsenoside Rg1 Inhibits Cell Proliferation and Induces Markers of Cell Senescence in CD34+CD38- Leukemia Stem Cells Derived from KG1α Acute Myeloid Leukemia Cells by Activating the Sirtuin 1 (SIRT1)/Tuberous Sclerosis Complex 2 (TSC2) Signaling Pathway.

BACKGROUND Clinical relapse in acute myeloid leukemia (AML) is associated with the reduced treatment response of leukemia stem cells (LSCs). This study aimed to investigate the effects of the ginseng derivative, ginsenoside Rg1 (Rg1), on CD34+CD38- LSCs derived from KG1a human acute myeloid leukemia cells. MATERIAL AND METHODS CD34+CD38- LSCs were isolated from KG1a human acute myeloid leukemia cells by cell sorting. CD34+CD38- KG1alpha LSCs were divided into the control group and the Rg1 group (treated with Rg1). The cell counting kit-8 (CCK-8) assay evaluated the proliferation of CD34+CD38- KG1alpha LSCs and flow cytometry studied the cell cycle. The mixed colony-forming unit (CFU-Mix) assay and staining for senescence-associated beta-galactosidase (SA-ß-Gal) evaluated cell senescence. Expression of sirtuin 1 (SIRT1) and tuberous sclerosis complex 2 (TSC2) were evaluated using Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS CD34+CD38- KG1alpha LSCs were isolated at 98.72%. Rg1 significantly reduced the proliferation of CD34+CD38- KG1alpha LSCs compared with the control group (p<0.05). Cells in the G0/G1 phase were significantly increased, and cells in the G2/M and S phase were significantly reduced compared with the control group (p<0.05). Rg1 significantly increased SA-ß-Gal and reduced CFU-Mix formation compared with the control group (p<0.05), significantly down-regulated SIRT1 expression in CD34+CD38- KG1alpha LSCs compared with the control group (p<0.05), and significantly reduced TSC2 expression in CD34+CD38- KG1alpha LSCs compared with the control group (p<0.05). CONCLUSIONS Rg1 inhibited cell proliferation and induced cell senescence markers in CD34+CD38- KG1alpha LSCs by activating the SIRT1/TSC2 signaling pathway.

[Effect of ginsenoside Rg_1 in delaying premature ovarian failure induced by D-gal in mice through PI3K/Akt/mTOR autophagy pathway].

The aim of this paper was to study the role of phosphoinositide 3-kinase(PI3 K), protein kinase B(Akt) and mamma-lian target of rapamycin(mTOR) in the inhibition of premature ovarian failure induced by D-galactose(D-gal) in mice model by ginsenoside Rg_1(Rg_1). Fifty-four female SPF BALB/c mice were randomly divided into PBS group, D-gal group, and Rg_1 group. In the D-gal group, D-galactose(200 mg·kg~(-1)·d~(-1)) was injected subcutaneously into the neck and back for 42 days. In the PBS group, an equal amount of phosphate buffered saline(PBS) was injected into the neck and back for 42 days. In addition to the therapy of D-gal group, Rg_1 group was given Rg_1(20 mg·kg~(-1)·d~(-1)) through intraperitoneal injection since the 15 th day for 28 days, at the same time, the D-gal group and the PBS group were also given an equal amount of PBS through intraperitoneal injection since the 15 th day for 28 days. After the treatment, the estrous cycle changes of the mice were detected, and the ovarian SA-ß-Gal staining was used to detect the changes of ovarian aging. Western blot was used to detect the changes in protein expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). Fluorescence quantitative PCR was used to detect the changes in mRNA expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). According to the findings, compared with the PBS group, the D-gal group began to show estrous cycle disorder in the 3 rd week,the ovarian SA-ß-Gal staining positive granulosa cells increased in the D-gal group, the expression of senescence marker P16~(INK4 a) increased, while the expression of autophagy signaling molecule LC3-Ⅱ decreased. After treatment with Rg_1, the positive rate of ovarian SA-ß-Gal staining in Rg_1 group decreased, the expression level of autophagy signaling molecule LC3-Ⅱ in Rg_1 group was higher than that in D-gal group, while the expression level of senescence marker P16~(INK4 a) was lower than that in D-gal group. Compared with the PBS group, the protein and mRNA expressions of PI3 K, Akt, mTOR and S6 k in the D-gal group were up-regulated, the protein expressions of Akt, mTOR and S6 k in the Rg_1 group were up-regulated, and the mRNA expressions of PI3 K and mTOR were up-regulated. After treatment with Rg_1, the protein expressions of PI3 K, Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group, while the mRNA expressions of Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group. The finding ssuggested that Rg_1 has the effect in delaying ovarian premature failure in D-gal-induced mouse models, and PI3 K/Akt/mTOR autophagy signaling pathways play an important role.

[Ginsenoside Rg_1 induces leukemia stem cell senescence via SIRT1/TSC_2 signal axis].

The aim of this paper was to investigate the effect of SIRT1/TSC_2 signal axis on leukemia stem cell senescence induced by ginsenoside Rg_1. CD34~+CD38~- leukemia stem cells(CD34~+CD38~-LSCs) was isolated by magnetic cell sorting(MACS) and divided into two groups. The control group cells were routinely cultured, 40 µmol·L~(-1) ginsenoside Rg_1 was added to the control group for co-culture in Rg_1 group. The effect of Rg_l to induce CD34~+CD38~-LSCs senescence were evaluated by senescence-associated ß-Galactosidase(SA-ß-Gal) staining, cell cycle assay, CCK-8 and Colony-Assay. The expression of senescence associated SIRT1, TSC_2 mRNA and protein was examined by Real-time fluorescence quantitative PCR(FQ-PCR) and Western blot. The results showed that the CD34~+CD38~-LSCs could effectively be isolated by MACS, and the purity of CD34~+CD38~-LSCs is up to(95.86±3.04)%. Compared with the control group, the percentage of positive cells expressed SA-ß-Gal in the Rg_1 group is increased, the senescence morphological changes were observed in the CD34~+CD38~-LSCs in the Rg_1 group. The proliferation inhibition rate and the number of cells entered G_0/G_1 phase in the Rg_1 group were increased, but the colony-formed ability was decreased, Rg_1 could significantly inhibit the proliferation and self-renewal ability of CD34~+CD38~-LSCs. The expression of SIRT1 and TSC_2 mRNA and protein were down regulated in the Rg_1 group compared with the control group. Our research implied that Rg_1 may induce the senescence of CD34~+CD38~-LSCs and SIRT1/TSC_2 signal axis plays a significant role in this process.

Synthesis characterization and cytotoxicity studies of platinum(II) complexes with reduced amino pyridine schiff base and its derivatives as ligands.

A series of reduced amino pyridine Schiff base platinum(II) complexes were prepared as potential anticancer drugs, and characterized by NMR, IR spectroscopy, elemental analysis, and molar conductivity. UV and CD results showed the binding mode between these compounds and salmon sperm DNA may be intercalation. The cytotoxicity of these complexes was validated against A549, Hela, and MCF-7 cell lines by MTT assay. Some complexes exhibited better cytotoxic activity than cisplatin against Hela and MCF-7 cell lines.

Ginsenoside Rg1 Attenuates Premature Ovarian Failure of D-gal Induced POF Mice Through Downregulating p16INK4a and Upregulating SIRT1 Expression.

BACKGROUND: Premature ovarian failure (POF) refers to pathological amenorrhea before 40 years. OBJECTIVE: To explore the regulatory effect of Rg1 on POF and clarify associated mechanisms. MATERIALS AND METHODS: POF mice were induced by injecting with D-galactose (D-gal, 200 mg/kg/- day). Mice were divided into phosphate buffered saline (PBS), D-gal (POF mice), D-gal/Rg1 group (POF mice administrating D-gal/Rg1). Weight growth rate and ovarian weight coefficient were measured. Serum estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), superoxide dismutase (SOD), catalase (CAT) levels were examined using ELISA. The status of follicle and corpus luteum was determined using hematoxylin-eosin (HE) staining. P16INK4a and silent- mating type information regulation-2 homolog-1 (SIRT1) were determined using western blotting and RT-PCR. RESULTS: Weight growth rate and ovarian weight coefficient of mice in D-gal group were significantly decreased than PBS group (p<0.05). Serum E2, LH, SOD, CAT levels were significantly decreased, FSH levels were remarkably increased in D-gal group than PBS group (p<0.05). Rg1 (D-- gal/Rg1 group) significantly increased weight growth rate and ovarian weight coefficient, enhanced E2, LH, SOD, CAT levels and decreased FSH levels than D-gal group (p<0.05). HE staining demonstrated normal follicle morphology/structure of mice in PBS group and decreased the number of follicles, obvious vacuolation of corpus luteum and increased atretic follicles of mice in D-gal group. Compared with D-gal group, the number of follicles was increased, luteal follicles were decreased in mice in D-gal/Rg1 group (p<0.05). Rg1 significantly (D-gal/Rg1) downregulated p16INK4a and upregulated SIRT1 expression in ovarian tissues of mice compared to the D-gal group (p<0.05). CONCLUSION: Rg1 could delay premature ovarian failure in D-gal induced POF mouse model through downregulating p16INK4a and upregulating SIRT1 expression.

Clinical characteristics and etiology of bacterial meningitis in Chinese children >28 days of age, January 2014-December 2016: A multicenter retrospective study.

OBJECTIVE: To explore the clinical characteristics and etiology of bacterial meningitis (BM) in Chinese children. METHOD: BM cases in children 28days to 18 years old were collected from January 2014-December 2016 and screened according to World Health Organization standards. Clinical features, pathogens, and resistance patterns were analyzed. RESULTS: Overall, 837 cases were classified into five age groups: 28 days-2 months (17.0%), 3-11 months (27.8%), 12-35 months (24.0%), 3-6 years (13.9%), and >6years (17.3%). Major pathogens were Streptococcus pneumoniae (S. pneumoniae, n=136, 46.9%), group B Streptococcus (GBS, n=29, 10.0%), and Escherichia coli (E. coli, n=23, 7.9%). In infants <3 months old, GBS (46.5%) and E. coli (23.3%) were most common; in children >3 months old, S. pneumoniae (54.7%), which had a penicillin non-susceptibility rate of 55.4% (36/65), was most frequent. The resistance rates of S. pneumoniae and E. coli to cefotaxime and ceftriaxone were 14.0%/40.0% and 11.3%/68.4%, respectively. All GBS isolates were sensitive to penicillin. CONCLUSIONS: The occurrence of BM peaked in the first year of life, while S. pneumoniae was the predominant pathogen in children >3months of old. The antibiotic resistance of S. pneumoniae was a concern.