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Objective:To prepare fenofibrate PEG2000-DSPE micelles in order to improve the solubility of fenofibrate, and study the oral pharmacokinetics of the micelles in SD rats. Methods:Fenofibrate PEG2000-DSPE micelles were prepared and characterized. The rats were administrated with fenofibrate PEG2000-DSPE micelles and fenofibrate suspension, respectively. The blood samples were collected from eye socket and determined by HPLC. The compartmental pharmacokinetics was analyzed by DAS software. Results:Fenofibrate PEG2000-DSPE micelles were prepared successfully. The mean particle size was (23. 40 ± 3. 62) nm, the drug loading and the entrapment efficiency was (97. 65 ± 3. 32) % and (1. 33 ± 0. 32) %, respectively. The mean plasma concentration-time curves of fenofibric acid were both in accordance with two-compartment mode after oral administration of fenofibrate PEG2000-DSPE micelles and fenofibrate suspension in rats. After the oral administration, AUC(0-24) and Cmax of fenofibrate PEG2000-DSPE micelles was respectively 7-fold and 14-fold higher than that of fenofibrate suspension [(61. 41 ± 5. 71)μg·h·ml-1 vs (8. 49 ± 0. 66)μg·h·ml-1, and (9.67±1.65) μg·ml-1 vs (0.71 ±0.09) μg·ml-1]. The relative bioavailability of fenofibrate PEG2000-DSPE micelles was 723. 3%. Conclusion:The bioavailability and absorption rate of fenofibrate are both increased by the micelles remarkably when com-pared with those of fenofibrate suspension after oral administration. The PEG2000-DSPE micelles served as drug carrier for oral delivery present promising application perspectives.
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OBJECTIVE:To study in vitro anticancer activity of conjugated linoleic acid-gemcitabine conjugate (CLA-GEM). METHODS:IC50 of different tumor cells (breast cancer MCF-7 cell,breast cancer MDA-MB-231 cell,lung cancer A549 cell, small cell lung cancer NCI-H446 cell,glioma C6 cell)were investigated after treated with different concentrations(0.001-100μmol/L)of CLA-GEM and gemcitabine(GEM)for 72 h;survival rates of MCF-7 cell were investigated after treated with above so-lution for 24,48 and 72 h. The dependence of 0.001-100 μmol/L CLA-GEM and GEM to nucleoside transporter was investigated (by IC50)through MCF-7 cells and MDA-MB-231 cells treated with nucleoside transporter inhibitors(NBMPR,100 μmol/L)and di-pyridamole(4 μg/ml). The change of MCF-7 cell cycle was investigated after treated with 1 μmol/L CLA-GEM and GEM for 24 h. RESULTS:Compared with GEM,IC50 of MCF-7,MDA-MB-231 and NCI-H446 cells became lower after treated with CLA- GEM (P0.05). Survival rate of MCF-7 cells de-creased significantly after treated with GEM for 48 h and CLA-GEM for 24 h. Survival rate of MCF-7 cells was the lowest,being 21% after treated with GEM for 72 h,while tumor cells were sacrificed by CLA-GEM completely. Compared with GEM or CLA-GEM,IC50 of MCF-7 and MDA-MB-231 cells increased significantly after treated with NBMPR,dipyridamole combined with GEM (P0.05). Compared with GEM,CLA-GEM could prolong 6% of S stage of MCF-7 cells (P<0.01). CONCLU-SIONS:Compared with GEM,CLA-GEM exhibits significant antitumor activity and rapid action,and it isn’t influenced by nucle-ic acid transportation.
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Objective To explore the relationship between intracellular concentration of arsenic trioxide (ATO) in ATO-resistant K562 cells (K562/AS2) and ATO resistance level.Methods The K562/AS2 cells were established by gradually increasing the concentration of ATO from the parental cell line,K562.Arsenic concentration was measured with atomic fluorescence photometry.Cell viability was assessed using MTT assay.Results At exposure to 1 μg/ml ATO for 24 h,48 h and 72 h,the arsenic concentration in the K562/S cells were all significantly higher than that in the K562/AS2 cells,(15.63± 0.42) μg/L vs 0 μg/L,(22.27±0.15) μg/L vs (3.51±0.12) μg/L and (24.31±0.21) μg/L vs (3.61±0.11) μg/L (P ＜ 0.05).With increasing concentration and the extension of incubation time,concentration of arsenic in cells was gradually increased (P ＜ 0.05),which increase quickly between 1 μg/ml and 2 μg/ml.The growth inhibition rate of K562/AS2 cells was also gradually increased (P ＜ 0.05),which increased quickly between 1 μg/ml and 2 μg/ml.Linear correlation analysis showed that when the K562/AS2 cells was exposed to ATO for 24 h,48 h and 72 h,respectively,the cell growth inhibition rates were positively correlated with the intracellular concentration of ATO.Conclusions Either increasing concentrations of ATO or prolonging the exposure time to ATO can increase intracellular concentration of ATO in ATO-resistant cells,and intracellular arsenic concentration is positively related to the cytotoxicity of ATO to K562/AS2 cells.Therefore,the sensitivity to ATO of ATOresistant K562 cells could be restored by increasing the intracellular concentration of ATO.
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Objective To observe the anti-tumor effect on human multiple myeloma cell lines U266 and KM3 with a combination of varinostat and melphalan in vitro.Methods The cell proliferation of U266 and KM3 was datected with MTT assay when treated them with vorinostat alone and melphalan alone,then calculate their IC50 values respectively.Fixed the concentrations of vorinostator melphalan,the cell proliferation was datected in combination with melphalan/vorinostat in seriesly concentrations by MTr assay.Then to calculate drug combination index(CI).Results The IC50 value of U266 was 5.0-7.5 μmol/L and that of KM3 was 2.5-5.0 μmol/L when treated by vorinostat alone,the IC50 value of U266 was 40-60 μmol/L and that of KM3 was 60-80 μmol/L treated by melphalan alone.When fixed the concentration of vorinostat(in U266 the concentration was 1.25 μmol/L,in KM3 the concentration was 1.0 μ mol/L),Synergism(CI＜0.9)was observed when the concentrations of melphalan were 20 μmol/L,40 μmol/L,60 μ mol/L and 80 μ mol/L in U266,40 μmol/L,60 μmol/L,80 μmol/L and 100 μmol/L in KM3;When fixed the concertration of melphalan (in U266,was 10 μmol/L,in KM3 was 20 μmol/L),synergism(CI＜0.9)was observed when the concentrations of vorinostat were 1.0 μmol/L,2.5 μmol/L,5.0 μmol/L and 7.5 μmol/L in U266,and 1.0 μmol/L,2.5μmol/L,5.0 μmol/L in KM3.An additive effect was observed with the czombination of vorinostat 7.5 μmol/L plus melphalan 20 μmol/L in KM3(CI=0.93).Conclusion Vorinostat had potential anti-myeloma effect alone,and had synergistic anti-tumor effect with melphalan in vitro.
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Objective To investigate the reversal effect of Topo Ⅱα-shRNA and Topo Ⅱβ-shRNA on Topo Ⅱ gene in K562/AS2 cells. Methods Three pieces of Topo Ⅱα-shRNA and three pieces of Topo Ⅱβ-shRNA were designed,synthesized and transfected into K562/AS2 cells by liposome. Expression level of Topo Ⅱα and Topo Ⅱβ mRNA were determined by real time fluorescence quantitative PCR. The expressions of Topo Ⅱα and Topo Ⅱβ protein were assayed with flow cytometer. Results After treated with Topo Ⅱα-shRNA or Topo Ⅱβ-shRN A for 24 hours,the expression level of Topo Ⅱα mRNA and Topo Ⅱβ mRNA protein in K562/AS2 cells decreased at most (78.22±0.01) %,(31.17±1.27) % (P <0.05),and (57.36±0.01)%,(23.98±1.22) % (P <0.05) respectively. Conclusion The expression of Topo Ⅱ gene can be down-regulated after infected with Topo Ⅱ-shRNA in K562/ AS2 cell line.
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Objective To investigate sequence variations of 12 miRNA genes in multiple myeloma(MM) in order to find whether sequence variations in miRNA genes are associated with tumorigenesis and discuss the clinical significance of MM associated with miRNA genes mutations. Methods The miRNA gene mutations in 20 cases of MM, 4 MM-derived cell lines and 20 controls were detected by the methods of polymerase chain reaction single stranded conformation polymorphism (PCR-SSCP) and silver staining technique. Both clinical features and laboratory results were analyzed simultaneously. Results The electrophoretic patterns showed a total of three variations in miR-19a, miR-19b and miRNA-335,which were observed in 3 MM cells (15 %, 3/20). We also found variations of miRNA-335 in MM-derived cell lines KM-3and RPMI8226. However, no sequence alteration in the miRNA genes was observed in our set of controls. One of the three MM patients died, and two of them were detected mutations at the terminal stage of the disease.Conclusion A relative high frequency of miRNA gene mutation was found in MM and MM derived cell lines, which suggests possibility of a main mechanism underlying tumorigenesis. And, detecting miRNA gene mutations in MM might be benefit to evaluate the progression and prognosis of disease.
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Objective To construct a recombinant adenovirus vector of Hairpin RNA specific for MRP1 gene and study its inhibition of MRP1 gene expression in K562/AS2 cell line resistant to AS_2O_3 (ATO). Methods A MRP1-specific hairpin RNA recombinant adenovirus vector was constructed and used to infected K562/AS2 cells. Expression level of MRP1 mRNA detected by real-time fluorescent quantitative PCR. MRP1 protein detected by flow cytometry. MTT method was used to detected the cytotoxicity of ATO and etoposide. Results MRP1 mRNA and protein expression level in K562/AS2 cells before and after the pAd-MRPl-shRNA adenovirus infection was (34.70±0.28 vs 4.19±0.03, P <0.05) and (26.40±0.16 vs 10.85±0.37, P<0.05), respectively. RR of K562/AS2 to arsenic trioxide and etoposide was (11.4078±0.3183 fold vs 1.6126±0.3015 fold, P<0.05) and (5.9141 ±0.0149 fold vs 1.7664±0.1038 fold, P <0.05), respectively. The reversal fold of ATO and etoposide was (7.2409±1.3668) and (3.3555±0.1886), respectively. Conclusion Successfully constructed pAd-EGFP-U6-shRNA-MRPl adenovirus vector, the vector of infection K562/SA2 cells can inhibit MRP1 gene expression and reverse the resistance of the ATO and etoposide.
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Objective To investigate the inhibitional effect of MRPI-shRNA on expression of MRP1gene in K56.2/AS2 cells resistant to arsenic trioxide. Methods Three pieces of MRPI-shRNA were designed,synthesized and transfected into K562/AS2 cells with lipesome. Expression level of MRPI mRNA were determined by real time fluorescent quantitative PCR. MRPI protein expression and intracellular accumulation of DNR were assayed with flow cytometry. Results After treated with MRPI-shRNA, the expression level of MRPI mRNA and MRPI protein in K562/AS2 cells decreased significantly(79.1±0.07) % and (62.48±0.86) %,respectively (P <0.05). The intracellular accumulation of DNR increased significantly(P < 0.05). Conclusion MRPI-shRNA can down-regulate the expression of MRPI gene in K562/AS2 cell line.
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Objective To evaluate the influence of tissue factor pathway inhibitor 2(TFPI-2)gene on the proliferation and invasion of K562.Methods The expression vector pcDNA3.0/TFPI-2 was transfected into human leukemia line K562 cells(K562-T)by using liposome,then the mRNA and protein TFPI-2 were detected by real-time RT-PCR and western blot separately.The growth curve and the colony-forming unit assay were used to measure the ability of cells growth and transwell chamber model was employed to test the ability of cell invasion in vitro.Results Expression of mRNA and protein of TFPI-2 was detected in transfected cells.The growth rate and self-replication ability of K562-T cells were lower than those of the two control groups obviously.The number of K562-T cells to traverse a Matrigel-coated membrane was dramatically decreased compared with that of non-expressing cells.Conclusion The gene of TFPI-2 can inhibit the growth,proliferation and invasion of the K562 cells.
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AIM: To establish a arsenic trioxide (As 2O 3 )-resistant leukemic cell line to explore the mechanism of resistance to As 2O 3, and the relationship between the resistant cell line and the multidrug resistance was also investigated. METHODS: The arsenic trioxide (As 2O 3 )-resistant leukemic cell line was established by exposing the cells to the increasing concentration of As 2O 3. MTT assay was used to detect the cytotoxicity. Cell cycle was detected by PI assay. Flow Cytometry was used to detect the P-glycoprotein on the surface of the cells, the intracellular concentration of DNR, and the immuetype of the cells. RESULTS: The cell doublings time and the cell cycle of the arsenic trioxide (As 2O 3 )-resistant leukemic cell line, K562/AS2, is similar to that of K562. The relative resistant fold of K562/AS2 to As 2O 3, DNR, VP16 and Ara-C was 7.4, 2.9, 3.8 and 1.1, respectively. The relative resistant fold of multidrug resistant cell line, K562/ A02, to As 2O 3, DNR, VP16 and Ara-C was 0.8?94?2.5 and 0.9, respectively. The fluorescence of the P-glycoprotein on the surface or of the DNR inside the cells detected was not significantly different between the K562 and the K562/AS2 cell lines. CONCLUSIONS: A cell line, K562/AS2, resistant to clinical achieving level (2 ?mol/L) of As 2O 3 has been established. The relative resistant fold of K562/ AS2 to As 2O 3 is about 7.4 fold to the parent K562 line sensitive to As 2O 3. Partial resistance of K562/AS2 to DNR and VP16 is observed , the mechanism of which is unrelated to the P-gp, the expression product of multidrug resistance gene 1 (mdr1).