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Objective To investigate the effects of chemotherapeutic drugs on ER-α expression and methylation in breast cancer cells.Methods Human breast cancer cells MCF-7(ER+,Luminal A) were induced by paclitaxel(PTX) and epirubicin(EPI) for more than 6 months,with an incremental dose,respectively.The expression and methylation status of ER-α in MCF-7 cells were detected before and after drug treatment.miRNAs with consistent expression changes in MCF-7 cells after two drugs' treatment were screened by microarray,and verified by quantitative PCR (qPCR).Targets of the most significantly down-regulated miRNA were analyzed by bioinformatics.miRNA inhibitor was transfected into MCF-7 cells,miRNA mimic was transfected into MCF-7/PTX and MCF-7/EPI cells,then ER-α and DNA methyltransferase 1 (DNMT1) expression were detected by Western blot,and ER-α methylation was detected by quantitative methylation-specific PCR (qMSP).Results PTX resistant MCF-7/PTX cell line and EPI resistant MCF-7/EPI cell line were established.Both drug treatments caused a decrease in ER-α protein expression and an increase in methylation levels,with up-regulation of DNMT1 and his tone deacetylase 1 (HDAC 1) expression.miRNAs with consistent expression changes in MCF-7 cells after drug treatments were screened and verified by qPCR,the most significant down-regulation among which was miR-148b.Bioinformatics analysis,and further confirmed by luciferase reporter gene assay (Luciferas) that DNMT1 was a direct target of miR-148b.miR-148b inhibitor induced decreased expression of ER-α and increased methylation level in MCF-7 cells,accompanied by increased expression of DNMT1;whereas miR-148b mimic caused an increased expression of ER-α and decreased methylation level in MCF-7/PTX and MCF-7/EPI cells,with a decreased expression of DNMT1.Conclusion Chemotherapeutic drugs (represented by PTX and EPI) induce aberrant miRNA expression in breast cancer MCF-7 cells,and down-regulate miR-148b further to attenuate the inhibition of DNMT1 expression,which promote,hypermethylation and down-regulation of ER-α.
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Objective@#To investigate the influences of bone marrow stromal cells, components of extracellular matrix and cytokine secreted by stromal cells on the chemotherapeutic sensitivity of acute lymphoblastic leukemia cells to cytosine arabinoside (Ara-C).@*Methods@#The co-culture model of acute lymphoblastic leukemia cell Sup-B15 and bone marrow stromal cell OP9 was constructed. Sup-B15 cells were cultured alone or co-cultured with OP9 cells, inactivated OP9 cells, the conditional medium (CM) of co-cultured OP9 cells and Sup-B15 cells, the CM of OP9 cells alone or Sup-B15 cells alone, respectively. The effects of different concentrations of Ara-C on the proliferation of each Sup-B1 cell group mentioned above were detected by cell counting kit-8 (CCK-8) method. The effects of different concentrations of Ara-C on the apoptosis of each group were detected by flow cytometry (FCM). The expressions of Bcl-2 protein in each group were detected by western blot.@*Results@#The results of CCK-8 test showed that the inhibitory efficiency of Ara-C was in a dose-dependent manner. With different concentrations of Ara-C treatment for 48 hours, the half maximal inhibitory concentrations (IC50) of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group were 0.510 and 0.339 μg/ml, respectively, significantly higher than 0.091 μg/ml of Sup-B15 cultured alone group (P<0.05). The IC50 of CM of Sup-B15 and OP9 co-cultured group was 0.204 μg/ml, significantly higher than 0.087 μg/ml of the CM of OP9 cultured alone group (P<0.05) and 0.097 μg/ml of the CM of Sup-B15 cultured alone group (P<0.05). The results of flow cytometry showed that with 0.10 μg/ml Ara-C treatment for 24 hours, the early apoptotic cell percentages of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group and Sup-B15 cultured alone group were (6.67±2.19) %, (8.95±3.04) % and (20.46±2.63) %, respectively. The early apoptotic cell percentages of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group were significantly lower than that of Sup-B15 cultured alone group (P<0.05). The early apoptotic cell percentages of the CM of Sup-B15 and OP9 co-cultured group, the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group were (11.16±2.97)%, (22.08±2.71)% and (19.25±1.57)%, respectively, the former two of which were significantly lower than the last one (P<0.05). The results of western blot showed that the relative expression levels of Bcl-2 protein of Sup-B15 cultured alone group, Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group, the CM of Sup-B15 and OP9 co-cultured group, the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group were 1.00±0.00, 1.53±0.03, 1.38±0.01, 1.26±0.05, 1.03±0.01 and 0.98±0.02, respectively. The expression levels of bcl-2 protein of three combined groups were significantly higher than that of Sup-B15 cultured alone group (P<0.05). while no statistically significant difference was observed between the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group (P>0.05).@*Conclusion@#Bone marrow stromal cell OP9, the components of bone marrow extracellular matrix and cytokine secreted by stromal cells are involved in the induction of the chemotherapeutic resistance of Sup-B15 cells to Ara-C.
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Objective: To explore the efficacy and safety of thalidomide for the treatment of delayed vomiting, induced by chemotherapy in cancer patients
Study Design: Randomized, double-blind controlled study
Place and Duration of Study: The Oncology Department of Affiliated Hospital of Xuzhou Medical University, Jiangsu Xuzhou, China, from January 2012 to January 2014
Methodology: A total of 78 cancer patients, who had delayed vomiting observed from 24 hours to 1 week after chemotherapy, were included in the study. Patients were divided in a treatment group [40 patients, 51.28%] and a control group [38 patients, 48.71%]. The treatment group received thalidomide at an oral dose of 100 mg per night; 50 mg was added daily up to a dose of 200 mg per night, if the curative effect was suboptimal and the medicine was tolerated. Both the treatment and the control groups received a drip of 10 mg azasetron 30 minutes before chemotherapy. The control group only proportions of antiemetic effects and adverse reactions were compared using the c2 test. Antiemetic effects and adverse reactions were assessed from Odds Ratios [OR] with 95% Confidence Intervals [95% CI]
Results: The effective control rate of delayed vomiting in the treatment group was significantly higher than that in the control group [c2=5.174, p=0.023]. No significant difference was found between the two groups in other adverse effects of chemotherapy. Karnofsky scores or the overall self-evaluation of the patients [p>0.05]
Conclusion: Thalidomide can effectively control the delayed vomiting of cancer patients receiving chemotherapy and the adverse reactions of the agent can be tolerated
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Objective:To investigate the effect on proliferation and apoptosis of T-cell leukemia cells by silencing NRP-1 ( Jurkat cells).Methods:The lentivirus plasmid which expresses NRP1 gene specific shRNA was constructed in our preliminary ex-perimental.We transfected the lentivirus plasmid to human T-cell Lymphoma cells.The proliferation of Jurkat cells different groups and effect on cell proliferation after chemotherapy drug EPI-treated were found by CCK-8 kit.The proliferation level and apoptosis rate of the cells were detected by flow cytometry and Annexin-V-FITC/PI method.Results:The proliferation level of NRP-1 /shRNA interference group was decreased significantly in 48 h,72 h,96 h,which was compared with the control groups.The apoptosis rate of the NRP-1/shRNA interference group was increased compared with control groups.The chemotherapy drug sensitivity of epirubicin ( EPI ) test results showed that EPI concentration was 0.025,0.05,0.1,0.2,0.4 μg/ml,the NRP-1/shRNA interference group of cell growth inhibition rate was increased,the corresponding control group difference had statistical significance(P<0.05).We choose the drug con-centration of the EPI IC50 for next experiments.NRP-1/shRNA interference group cell apoptosis rate increased significantly after induction,compared with the control groups difference was statistically significant ( P<0.05 ).Compared with control group, the expression level of Bcl-2 protein was decreased and the expression level of bax protein was increased significantly after EPI induction.The percentage of cells at G0/G1 phase increased significantly,while those at S phase decreased significantly.Conclusion:Plasmid shRNA-NRP1 inhibited the expression of NRP1 in Jurkat cells and decreased the proliferation level of Jurkat cells and promote their apoptosis and enhance their drug sensitivity;the molecular mechanism may relate to down-regulation of Bcl-2 and up-regulation of Bax.and arrested the cell cycle at G0/G1 phase.
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Objective:To construct GST-tagged human NRP-1 fusion protein expression vector and induce its expression in Escherichia coli ( E.coli) ,then carry on inclusion body refolding and purification so as to obtain GST-NRP-1 fusion protein.Methods:NRP-1 gene was amplified by RT-PCR and inserted into pCR-blunt vector.Then the reconstructed plasmid was inserted into prokaryotic expression vector pGEX-4T-1.The constructed pGEX-4T-1-NRP-1 expression vector was transformed into BL21 cells and induced by i-sopropyl-β-D-thiogalactoside ( IPTG).Bacterial bodies were disrupted by sonication.Then the soluble fraction of fusion proteins were verified by Western blot and purified by Glutathione Sepharose 4B after inclusion body refolding.Results: The NRP-1 gene fragment was amplified by RT-PCR and inserted into pCR-blunt vector.Fusion protein expression vector pGEX-4T-1-NRP-1 was constructed suc-cessfully.After transformation, GST-NRP-1 expression vector was detected in BL21 cells and obtained purifying protein after refolding.Conclusion:The plasmid GST-NRP-1 was constructed successfully and laid basis for subsequent studies.
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Objective To investigate the effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) combined with recombinant bovine basic fibroblast growth factor (rb-bFGF) in the treatment of oral ulcer caused by chemotherapy.Methods 108 patients of oral ulcer caused by chemotherapy were randomly divided into two groups.54 cases in the control group were treated with cydiodine buccal tablets at first,then received the aerosol treatment which was prepared by mixing gentamicin,dexamethasone,2 ％ lidocaine and physiologic saline,three times per day.54 cases in the treatment group firstly received the gargle which was prepared by mixing rhGM-CSF,dexamethasone and physiologic saline,then were treated with rb-bFGF by spraying on the oral ulcer surface,three times per day.Results The effective rate of the treatment group was 96.30 ％ (52/54),which was significantly higher than that of the control group [64.81％ (35/54)],there was a significant difference between the two groups (x2 =17.08,P ＜ 0.05).Conclusion The effect of rhGM-CSF combined with rb-bFGF in the treatment of oral ulcer caused by chemotherapy is very significant.
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Objective To enrich breast cancer stem cells of breast cancer cell lines MCF-7 and MDA-MB-231 through culturing mammospheres,and to detect the expression differences of gene of breast cancer stem cells makers.Methods The MCF-7 and MDA-MB-231 cell lines were cultured by serum and serum-free medium.The proportion of CD44+/CD24-and CD133+ cancer stem cells was measured in cells derived from mammosphere cells or monolayer culture cells by flow cytometry,and the expression of CD44,CD24,CD133,ALDH3A1,ABCG2 and CXCR4 mRNA were detected by RT-PCR.Results Flow cytometry analysis indicated that the CD44+/CD24-low proportion of the MCF-7 mammosphere cells was higher than its adherent culture cells (P ＜ 0.05),and CD133+ proportion had no difference between them (P ＞ 0.05).However,the CD44+/CD24-/low proportion of the MDA-MB-231 mammosphere cells was lower than its adherent culture cells (P ＜ 0.05),while CD133+ proportion was significantly higher than its adherent cultured cells (P ＜ 0.05).RT-PCR analysis suggested that the expression of CD44 and ABCG2 increased obviously in MCF-7 microspheres (P＜ 0.05),and the expression of CD24,CD133,ALDH3A1 and CXCR4 had no significant difference between the mammosphere cells and adherent culture cells (P ＞ 0.05).The CD24,CD133,ABCG2,ALDH3A1 and CXCR4 increased obviously in MDA-MB-231 microspheres.On the contrary,the CD44 decreased (P ＜ 0.05).The expression of CD44 and CD24,CD133 and ALDH3A1 had significant differences in the microspheres of MCF-7 and MDA-MB-231 cells (P ＜ 0.05).Conclusion The related cancer stem cells gene expression is different in the microspheres of human breast cancer cell line,which indicates that the different subtypes of breast cancer may be derived from different origins.
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Objective To explore the therapeutic effects and adverse reactions of thalidomide in the treatment of peri-chemotherapy nausea and vomiting of cancer patients.Methods Total of 70 patients were randomly divided into two groups:the treatment group (38 cases) and the control group (32 cases).The treatment group was treated with thalidomide (oral administration at a dose of 100 mg per night,then dose can be added by 50 mg until the top dose of 200 mg per day).The original will be maintained if they cannot be tolerate of extensive dose.The treatment group was also injected 2mg tropisetron in 30 minutes before chemotherapy.The control group was only injected same dose tropisetron.All cases were examined antiemetic effects and evaluated adverse reactions.Results Nausea and vomiting control rates were 89.5 ％ (34/38) and 68.8 ％ (22/32) respectively in the treatment group and control group respectively with significant difference.The adverse reactions were similar between the two groups.Conclusion Thalidomide joint tropisetron can effectively control the peri-chemotherapy nausea and vomiting,and the adverse reactions can be acceptable.It could improve further indications of the drug.