Establishment of real-time observational models of apoptosis induced by the aroma-tase inhibitor letrozole in breast cancer cells / 中国肿瘤临床

Objective: To construct an aromatase-overexpressing breast cancer cell model and observe the real-time apoptosis of breast cancer cells induced by the aromatase inhibitor, letrozole. Methods: The lentivirus-mediated gene transfection method was used to construct the MCF-7-VC3AI and ZR7530-VC3AI cell lines,which stably expressed the apoptotic fluorescent indicator protein VC3AI.Simultaneously,letrozole-induced apoptosis models of the MCF-7-VC3AI and ZR7530-VC3AI breast cancer cell lines were also constructed.Real-time quantitative PCR(qPCR)and Western blot methods were used to detect the mRNA and protein levels of aroma-tase in the cells.Cell proliferation ability was measured using MTT.The proliferation of cells in vitro under testosterone,estradiol,or letrozole combined with testosterone treatments were also observed.Results:qPCR results showed that the expression of the aroma-tase mRNA was significantly higher in both the MCF-7 and ZR7530 cell models when compared to the MCF-7-VC3AI and ZR7530-VC3AI cell models.Western blot results showed that the expression of the aromatase protein was significantly increased in both cell models. MTT assay results showed that the proliferation of a cell model could be promoted by testosterone and estrogen stimulation.Under 100 nmol/L testosterone,the proliferation rate of over-expressed aromatase MCF-7-VC3AI cells was about 1.2 times than that of the control group(P<0.01)and the proliferation rate of ZR7530-VC3AI cells was about 1.5 times than that of the control group(P<0.01). However,letrozole inhibited the proliferation induced by testosterone in a dose-dependent manner.Under the effect of letrozole at 10 μmol/L,the proliferation rate of over-expressed aromatase MCF7-VC3AI cells was 80% of the control group(P<0.05),while the prolifer-ation rate of over-expressed aromatase ZR7530-VC3AI cells was 68% of the control group(P<0.05).Conclusions:The successful estab-lishment of cell models that can detect letrozole-induced apoptosis provides an important foundation for further investigating the mechanism of letrozole.

The role of nuclear factor-kappa B in tamoxifen-resistance of breast cancer / 肿瘤

Objective: To explore the role of nuclear factor-kappa B (NF-κB) signaling pathway in tamoxifen resistance of breast cancer cells. Methods: MTT assay was used to detect the inhibitory effect of 0-25 μmol/L tamoxifen on the proliferation of hormone recept or-positive breast cancer MCF7 and MCF7R/LCC9 cells. Real-time fluorescence quantitative PCR and Western blotting were used to detect the levels of NF-κB mRNA and its inhibitory subunit α (IKBα) protein in these two breast cancer cells, respectively. MCF7-GC3AI cells were infected with the lentivirus carrying the mutant plasmid pCDH-CMV-IKBα (S32A, S262A), pCDH-CMV-IKKβ (S177E, S181E) and the empty vector pCDH-CMV-Vector. Then the inhibitory effect of tamoxifen in combination with NF-κB signaling pathway inhibitor ACT001 on proliferation of NF-κB signaling pathway-inactivated, -activated and normal MCF7-GC3AI cells was detected by MTT assay. The apoptosis of MCF7-GC3AI cells in each group was observed under a fluorescence microscope, and the clone formation ability of these cells was detected by colony formation assay. Results: MCF7R/LCC9 cells were resistant to tamoxifen as compared with MCF7 cells (P < 0.01). The expression level of negative regulator IKBα of NF-κB signaling pathway in MCF7R/ LCC9 cells was decreased (P < 0.01), and the mRNA level of NF-κB was increased (P < 0.01) as compared with MCF7 cells. After treatment with tamoxifen for 48 h, the proliferation inhibitory ability and apoptosis rate of MCF7-GC3AI-IKKβ cells were decreased (P < 0.01, P < 0.05), and the cell colony formation ability was increased (P < 0.05) as compared with MCF7-GC3AI-Vector cells. But the proliferation inhibitory ability and apoptosis rate of MCF7-GC3AI-IKBα cells were increased (P < 0.05, P < 0.01), and the cell colony formation ability was decreased (P < 0.05) after tamoxifen treatment for 48 h. ACT001, an inhibitor of NF-κB signaling pathway could increase the cytotoxicity of tamoxifen in MCF7R/LCC9 cells (P < 0.01). Conclusion: NF-κB signaling pathway maybe play an important role in the development of tamoxifen resistance in hormone receptor-positive breast cancer cells.

Investigation of the Liver-targeting Properties of Quercetin-loaded PLGA-TPGS Nanoparticles in HCa-F Cell-bearing Mice / 中国医科大学学报

Objective The aim of this study was to investigate the distribution and liver-targeting properties of quercetin (QT)/coumarin 6 (C6)-loaded polylactic-co-glycolic acid-D-α-tocopherol polyethylene glycol 1000 succinate (PLGA-TPGS) nanoparticles (QCPTN) in a hepatocarcinoma ectopic transplantation solid tumor model using HCa-F cell-bearing mice.Methods The QT concentrations in biological samples were determined using reverse phase-high performance liquid chromatography (RP-HPLC) analysis.After intravenous administration to mice in the QCPTN and QTS groups,the QT concentration in plasma and in different tissues was simultaneously analyzed at the different time points.Detection indexes (relative targeting efficiency,Re;targeting efficiency,Te) and fluorescence inversion microscopy images of the frozen tissue (liver,solid tumor,spleen,lungs,kidneys,and heart) slices were used for quantitatively and qualitatively evaluating the liver-targeting properties of QCPTN in solid tumor-bearing mice.Results Te of the QCPTN group in the plasma,liver,solid tumor,spleen,lungs,kidneys,and heart were all greater than 3,indicating that the area under the concentration-time curve (AUC) of liver was more than three times that of the plasma and other organs.Fluorescence inversion microscopy images showed that the green fluorescence of QCPTN was mostly observed in the liver.Conclusion Using HCa-F cell-beating mice,QCPTN was found to have better in vivo liver-targeting properties in hepatocarcinoma ectopic transplantation solid tumor.

CLONING OF ZNRD1 AND EFFECT OF ITS ANTISENSE VECTOR ON ACCUMULATION OF ADRIAMYCIN IN DRUG-RESISTANT GASTRIC CANCER CELLS / 解放军医学杂志

The aim of this study was to construct antisense of ZNRD1 encoding gene, and to transfect it into SGC7901/VCR cells, to seek measures to overcome multidrug resistance of gastric cancer cells. ZNRD1 cDNA amplified by PCR was confirmed by DNA sequencing, and then inserted into the multiple cloning site of the expressing vector pcDNA3 1 + with molecular cloning technique. The recombinant vector was identified by endonuclease digestion. Antisense recombinant vector was transfected into SGC7901/VCR cells using lipofectamine. Flow cytometric analysis was used to detect adriamycin (ADM) accumulation in SGC7901/VCR cells. The results showed that a fragment was obtained by PCR, and its sequence was consistent with ZNRD1 cDNA reported in the literature. After antisense recombinant vector having been transfected into SGC7901/VCR cell, ADM concentration in such cells was increased. The above results indicated that the antisense vector ZNRD1 could enhance the intracellular accumulation of ADM in SGC7901/VCR cells, which might be of potential treatment value.