Background and purpose:Breast cancer is one of the most common carcinoma among female patients with high mortalities. Drug-resistance is the major reason that leads to chemotherapy failure in clinical practice. MCF-7/ADR is a multi-drug resistant cell line that was established on the basis of breast cancer cell line MCF-7. This research aimed to investigate the anti-apoptotic effect of c-fos in resistant breast cancer cell MCF-7/ADR, and to compare with its sensitive counterpart MCF-7.Methods:Doxorubicin with various concentrations was used to treat MCF-7 as well as its MDR- counterpart MCF-7/ADR. The growth inhibitory rate of MCF-7 and MCF-7/ADR wasdetermined by MTT assay. Additionally, RT-PCR was used to test the expression of P-gp and c-fos mRNA in MCF-7 and MCF-7/ADR; The expression of c-fos mRNA was detected by RT-PCR after 3 μmol doxorubicin treatment;We further established cell lines that stably interfered with c-fos, named MCF-7/ADR/si-fos-8B, MCF-7/ADR/si-fos-3D. Flow cytometry and MTT assay were used to investigate the apoptosis rate and inhibitory rate in these above cells under the treatment of 5-FU, CDDP or γ-radiation. At last, RT-PCR and Western blot analysis were used to detect the expression of bax, bcl-2, puma, p53.Results:The expression of c-fos and P-gp (MDR-1) was up-regulated in MCF-7/ADR, compared with its sensitive counterpart MCF-7. Additionally, the resistant fold of MCF-7/ADR to doxorubicin was nearly 40; The expression of c-fos was gradually up-regulated after 3 μmol doxorubicin treatment; The sensitivity to drugs (5-FU and CDDP) was increased after c-fos interference while the apoptosis rate was also increased after 5-FU, CDDP and γ-radiation treatment. RT-PCR and Western blot analysis indicated that up-regulation ofbax,puma,p53 after c-fos interference while the expression of bcl-2 was down-regulated.Conclusion:c-fos may act as an anti-apoptotic protein in resistant breast cancer cell line MCF-7/ADR by regulating the expression of apoptosis related proteins, and may play a vital role in the formation of multi-drug resistance phenotype.

Aim To study the inotropic action of DMA in normal rat isolated hearts and papillary muscle and search for its primary mechanism.Methods With Lansendorff-perfusion and isolated papillary muscle perfusion,cardiac performance and the systolic function of papillary muscle were measured to estimate the inotropic action of DMA;L-calcium channel blocker and sodium calcium exchanger(NCX)inhibitor were used to search for its primary mechanism.Results ①(1~20)?mol?L-1 DMA exerted a positive inotropic action in normal rat isolated hearts,that is in Langendorff-perfused hearts,DMA enhanced cardiac performance,i.e.LVSP-LVDP,+dp/dtmax,-dp/dtmax significantly(P

Aim To study the mechanism of synergistic antitumor of EBB and doxorubicin in doxorubicin-resistant MCF-7/ADR breast carcinoma cells.Methods The antitumor activity of doxorubiein alone and its combination with EBB were measured by MTT assay in MCF-7/ADR and MCF-7cells. The rate of doxorubicin-induced apoptosis and the protein and mRNA levels of P-glycoprotein(P-gp) were determined in MCF-7/ADR treated with EBB by flow cytometry (FACS), respectively.Laser scanning confocal microscopy was used to detect the intracellular accumulation of drug in EBB-treated MCF-7 and MCF-7/ADR cells.Results EBB had antitumor effects for MCF-7 and MCF-7/ADR.It could potentiate the antitumor effect of dororubicin with CDI of 0.73 and 0.49 for MCF-7 and MCF-7/ADR,respectively.EBB and doxorubicin acted synergistically in elevating apoptosis of MCF-7/ADR and downregulating the expression of P-gp in a dose-dependent manner in MCF-7/ADR.EBB restored the intracellular accumulation of doxorubicin in MCF-7/ADR cells in a dose-dependent manner.After pretreatment with EBB for 24 h and 48 h,the intracellular accumulation of doxorubicin and Rh123 was obviousely restored in MCF-7/ADR cells compared with control in a time-dependent manner.Conclusion EBB is a potential agent which has strong inhibitory effect on both multidrug resistant cells and their parental cells.EBB can significantly potentiate the antitumor effects of dororubicin in MCF-7/ADR cells by blocking the function of P-glycoprotein and inhibiting the expression of P-glycoprotein.

Background and purpose:Bispecific antibody is a special pattern of genetic engineering antibody.It possesses 2 antigen binding sites, one directed at the effecter cells and the other at the target cells which are always the tumor cells.This enables the effecter cells to recruit to the targets efficiently and exert cytotoxic effect.The experiment was aimed to construct the expression vector of anti-CD3?anti-CD19 bispecific antibody, purification and analyzing the activity of the diabody.Methods:Applyed PCR and overlap PCR to construct the expression vector of anti-CD3?anti-CD19 bispecific antibodies, transformed it into E.coli 16C9 for expression.Purified the diabody by His-tag affinity chromatography and confirmed by 12% SDS-PAGE and Western blot.Antigen binding activity of the diabody was analyzed by FACS.Results:The DNA sequence of the expression vector containing anti-CD3?anti-CD19 fragment was comfirmed.The yield of purified diabody was up to 5 mg/L.The molecular weight of diabody was correctly indicated by SDS-PAGE and Western blot.The diabody could specifically bind to CD19+ Raji cells and CD3+ Jurkat cells while competitively inhibit binding of HIT3a and HIT19a to the cells.Conclusion:Successfully constructed the expression vector of anti-CD3?anti-CD19 bispecific antibodies, the diabody acquired by the method could be produced with high level expression and also had high specific affinity with CD19+ Raji cells and CD3+ Jurkat cells.

Objective:To prepare and characterize specific and discrepant mouse hybridoma antibodies on membrane of HL60 and HL60/ADR cell lines.Methods:BALB/c mice were immunized by subtractive immunization induced Cp(Cyclophosphamide).McAbs were prepared by hybridoma technique,screened and detected by FACS and LSCM.Results:51 candidates and discrepant antibodies were found,and one of them (5F6) was purified and identified.Conclusion:Combination of SI with discrepant screening method should facilitate the preparing and identifying discrepant McAbs for identifying antibodies that can distinguish the differences in proteins expressed in HL60 and HL60/ADR,which is a significative and potential method in the research and target therapy associated drug-resistance.

Background and purpose:Multidrug resistance(MDR)is one of the major causes of progressive breast cancer chemotherapy failure.One of the major mechanisms of MDR is the overexpression of P-glycoprotein (P-gp).Therefore,the identification of novel agents which can inhibit the drug transporter function of P-gp or its expression is of utmost interest in cancer research.The aim of this study was to explore the antitumor and reversal effect of PHⅡ-7,natural products from traditional Chinese medicine(TCM).Methods:The cytotoxicity of PHⅡ7 alone and combined application of PHⅡ-7 and adriamycin(ADR)on breast cancer cells were determined using MTT assay.Annexin V–FITC/PI apoptosis detection kit was used to observe the apoptosis-inducing effect of PHⅡ-7 in MCF-7 and MCF-7/ADR cells.The effect of PHⅡ-7 on mdr1 mRNA was determined by reverse transcription PCR and real time PCR,flow cytometer was used to measure the intracellular ADR accumulation.Results:PHⅡ-7 alone inhibited cell growth of MCF-7 and MCF-7/ADR cells with the IC 50 (6.07?0.85),(5.51?1.22)?mol/L,respectively when combined with ADR,PHⅡ-7 enhanced the cytotoxicity of ADR toward MCF-7/ADR cells.In addition,PHⅡ-7 induced apoptosis both on MCF-7 and MCF-7/ADR cells;PHⅡ-7 reversed the drug resistance to ADR in MCF-7/ADR cells by inhibiting mdr1 mRNA transcription and increasing the intracellular ADR accumulation.Conclusion:PHⅡ-7 displayed significant anti-proliferative and apoptosis-inducing effect on sensitive and multidrug resistant breast cells in vitro.PHⅡ-7 reversed effectively MDR by blocking the drugs to be pumped out by inhibiting P-gp expression and function pathway.

Objective: To investigate the effects of hesperetin on fine particulate matter (PM_2.5) induced apoptosis in H9c2 cells and related mechanisms. Methods: H9c2 cells were divided into 4 groups: control group (cells were cultured without intervention), PM_2.5 group (cells were treated with 800 µg/ml PM_2.5), hesperetin group (H group, cells were treated by 40 µmol/L hesperetin for 1 h at 37 ℃), and hesperetin+PM_2.5 group (H+PM_2.5 group, cells were pretreated with hesperetin before PM_2.5 treatment). Cells were cultured for corresponding interval. Apoptotic cells were detected by Annexin Ⅴ-FITC/PI apoptosis detection kit and Hoechst staining. The intracellular reactive oxygen species (ROS) levels were measured by DCFH-DA Fluorescence Probe and mitochondrial membrane potential (MMP) was detected with JC-1 staining, respectively in these groups. Apoptotic related protein and phosphorylated MAPK expression levels were determined by Western blot. Results: (1) Flow cytometry results showed that the apoptosis rate of PM_2.5 group ((48.94±3.20)%) was significantly higher than that of control group ((8.13±1.40)%, P<0.01), which was significantly reduced in H+PM_2.5 group ((34.80±2.21)%) (P=0.003 2 vs. PM_2.5 group, P<0.01 vs. control group). The number of Hoechst 33258 positive apoptotic cells was distinctly less in H+PM_2.5 group than in PM_2.5 group. (2) The ROS levels was significantly higher in PM_2.5 group ((49.69±5.05)%) than in control group (10.57±1.33)%, P<0.01), which was significantly reduced in H+PM_2.5 group ((35.08±3.90)%) (P=0.000 2 vs. PM_2.5 group, P<0.01 vs. control group). (3) Green fluorescence indicating the JC-1 monomer form, which represented MMP loss of H9c2 cells, was significantly higher in PM_2.5 group ((20.28±4.69)%) than in control group ((10.50±2.72)%, P<0.01), which was significantly decreased in H+PM_2.5 group ((13.41±2.89)%) (P<0.01 vs. PM_2.5 group, P=0.029 4 vs. control group). (4) The expression levels of Bcl-2 protein of H9c2 cells was lower in PM_2.5 group ((76.94±4.52)%) than in control group (100%, P=0.000 9), which was significantly upregulated in H+PM_2.5 group ((92.95±6.82)%) (P=0.027 5 vs. PM_2.5 group, P=0.15 vs. control group). The expression levels of cleaved caspase-3 protein of H9c2 cells was significantly higher in PM₂.5 group ((243.98±17.94)%) than in control group (100%, P=0.000 2), which was significantly downregulated in H+PM_2.5 group ((200.45±4.31)%) (P=0.015 vs. PM_2.5 group, P<0.01 vs. control group). (5) The expression levels of phosphorylated p38 MAPK protein of H9c2 cells was higher in PM_2.5 group ((118.90±4.78)%) than in control group(100%, P=0.002 7), which could be significantly downregulated in H+PM_2.5 group ((103.30±1.27)%) (P=0.01 vs. PM_2.5 group, P=0.05 vs. control group). The expression levels of phosphorylated ERK protein of H9c2 cells was higher in PM_2.5 group ((163.50±4.98)%) than in control group (100%, P<0.01), which was significantly downregulated in H+PM_2.5 group ((139.10±2.72)%) (P=0.001 6 vs. PM_2.5 group, P<0.01 vs. control group). Conclusions Hesperetin protects H9c2 cells from PM_2.5 stimulation through reducing oxidative stress and protecting mitochondrial function, regulating the expression of apoptotic associated proteins as well as MAPK signal pathway, thus inhibiting H9c2 cells apoptosis.

AIM: To investigate the effect and mechanism of ursolic acid on high glucose and high-fat injury of human aortic endothelial cells. METHODS: MTT method was used to select the appropriate injury concentration of high glucose and sodium palmitate and UA pre incubation concentration. The levels of NO and ROS, the release rate of lactate dehydrogenase and the activities of antioxidant enzymes including total superoxide dismutase, catalase and glutathione peroxidase were detected by kit. The expression of endothelial nitric oxide synthase, intercellular adhesion molecule, vascular cell adhesion molecule caspase-1 and GSDMD were detected by Western blot. The protective effect of UA on HAEC was observed. Hoechst33342 combined with PI fluorescence staining was used to detect the whole state of cell membrane to explore the occurrence of pyroptosis. RESULTS: Pre-incubation with UA (1 and 5 μmol/L) could reduce the damage of HAEC caused by high glucose and high fat (30 mmol/L Glu + 0.1 mmol/L SPA), enhance HAEC activity, increase NO release and eNOS protein expression, alleviate oxidative stress injury, reduce the protein expression of adhesion molecules and reduce the occurrence of pyroptosis. CONCLUSION: UA may reduce the damage of endothelial cells by inhibiting the oxidative stress response and the occurrence of pyroptosis induced by high glucose and high fat.