New 4-imino-4H-Chromeno[2,3-d]Pyrimidin-3(5H)-Amine: Synthesis, Cytotoxic Effects on Tumoral Cell Lines and in Silico ADMET Properties

The synthesis of new 4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine in four steps including one step under microwave dielectric heating is reported. The structural identity of the synthesized compounds was established according to their spectroscopic analysis, such as FT-IR, NMR and mass spectroscopy. These new compounds were tested for their antiproliferative activities on seven representative human tumoral cell lines (Huh7 D12, Caco2, MDA-MB231, MDA-MB468, HCT116, PC3 and MCF7) and also on fibroblasts. Among them, only the compounds 6c showed micromolar cytotoxic activity on tumor cell lines (1.8 50 50 > 25 μM). Finally, in silico ADMET studies ware performed to investigate the possibility of using of the identified compound 6c as potential anti-tumor compound.

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

Potential roles of EZH2, Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P < 0.01), while mi R-203 level was significantly lower in HCC tissues(P < 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P < 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P < 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P < 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.

MiR-25-3p attenuates the proliferation of tongue squamous cell carcinoma cell line Tca8113

Objective:To investigate the effects of miR-25-3p on the occurrence,development and proliferation of tongue squamous cell carcinoma cells.Methods:To establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant reiroviral vector-mediated gene transfer method.The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide(MTT)and cell colony formation assays.eyclnD1,p21^(cipt)and p27^(kipt)mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR.cyclinD1,p21^(cipt),p27^(kipt),AKT,p-AKT,FOXOt and p-FOX01 expressions in the transfected Tca8113 were detected by western blot analysis.In addition,miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR.Results:Quantitative PCR showed that mitt-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue.MTT and cell colony formation assays showed that after miR-25-3p overexpression,the proliferation of transfected Tca8113 was obviously attenuated.Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression.p21^(cipt)and p27^(kipt)expressions were upregulated,while cyclinD1,AKT,FOXO1 expressions were downregulated,and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells.Conclusions:MiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression,playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.

Contragestazol (DL111-IT) inhibits proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo

Aim： To evaluate the antiproliferative activity of contragestazol （DL111-IT） on the human prostate cancer cell line PC3 in vitro and in vivo and to elucidate its potential molecular mechanisms. Methods： The cell killing ability of DL111-IT was measured by the 3-（4,5-dimethylthia-zol,2-yl）-2,5-diphenyltetrazolium bromide （MTT） reagent assay method and the tumor xenograft model. The cell cycle was analyzed by flow cytometry and protein expression, including retinoblastoma （pRb）, cyclin-dependent kinase 4 （CDK4） and cyclin D 1, was detected by Western blotting. Results： DL111-IT exhibited high efficiency on cell growth inhibition of the human androgen-independent prostate cancer cell line PC3. The drug concentration that yielded 50 % cell inhibition （IC50 value） was 9.9 mg/mL. In the PC3 tumor xenograft study, DL111-IT （1.25 mg/kg-20.0 mg/kg） given once a day for 10 days significantly inhibited tumor growth, with the inhibition rate ranging from 21% to 50 %. Flow cytometric analysis indicated that DL111-IT could cause GI arrest in the PC3 cell line, but not apoptosis. DL111-IT enhanced pRb expression and down-regulated CDK4 and cyclin D 1 expression, suggesting that cell cycle regulation might contribute to the anticancer property of DL 111- IT. Conclusion： DL111-1T inhibits the proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo by a cell cycle regulation pathway.

Inhibitory effect of a new gossypol derivative apogossypolone (ApoG2) on xenograft of human prostate cancer cell line PC-3

Objective: To investigate the inhibitory effect of apogossypolone (ApoG2) on prostate cancer cell line PC-3 in vivo, and explore its mechanism. Methods: The models of transplantation tumors in Balb/c nu/nu mice were established via subcutaneous injection of PC-3 cells and the tumor-transplanted mice were divided into 4 groups: control group and three ApoG2 treatment groups, with 10 mice in each group. Volumes of the tumor were estimated every 2 d and the morphology of tumor tissues was observed. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and caspase-8 in tumor tissues. Results: ApoG2 (2.5 mg/kg-10 mg/kg) given intraperitoneally once a day can obviously inhibit the growth of subcutaneous prostatic carcinoma implant. The tumor volume decreased obviously when the treatment dosage was bigger than 5.0 mg/kg (P<0.01). Meanwhile, ApoG2 decreased the expression of PCNA and CD31, and enhanced the expression of caspases-3, caspase-8 in tumor tissues. Conclusion: ApoG2 exert an inhibitory effect on prostatic carcinoma possibly by inducing apoptosis and inhibiting tumor angiogenesis.

The U937 cell line induced to express CD14 protein by 1,25-dihydroxyvitamin D3 and be sensitive to endotoxin stimulation

BACKGROUND: CD14 was first described as a differentia- tion antigen on the surface of myeloid lineage cells. It acts as a glycosylphosphatidylinositol ( GPI)-anchored receptor for the complex of lipopolysaccharide (LPS) and plays a key role in the activation of LPS-induced monocytes. The purpose of this study was to observe the expression of CD14 protein and its gene in the human U937 promonocytic cell line when these cells were exposed to 1,25-dihydroxyvita- min D3 ( VitD3 ) and investigate their sensitivity to endo- toxin stimulation. METHODS: U937 cells were exposed to (0.1 μmol) VitD3 for 24 hours and were induced to express the CD14 mRNA gene and CD14 protein, then their responses were observed when they were stimulated with different concentrations of LPS for different time. RESULTS: The U937 cells induced by VitD3 were found to stably express CD14 mRNA and CD14 protein. And CD14 protein enhanced the sensitivity of U937/CD14 cells to li- popolysaccharide ( LPS ) stimulation. NF-ΚB in U937/ CD14 cells can be activated with low concentration of LPS (1 ng/ml-10 ng/ml), the TNF-α mRNA gene was in- duced , and then TNF-α was produced and released into the supernatant of culture. CONCLUSION: VitD3 can induce U937 cell to express the CD14 gene and CD14 protein and enhance the response of this type of cells to LPS stimulation.

THE DIFFERENTIATION OF HUMAN GASTRIC ADENOCAR-CINOMA CELL LINE MGc80-3 INDUCED BY DIBUTYRYL cAMP IN VITRO

For providing some experimental basis in establishing malignant phenotypic reversed indexes of gastric carcinoma cells, human gastric adenocar-cinoma cell line MGc80-3 was induced by dBcAMP in vitro to appraise the effect of gastric carcinoma cell differentiation by chemical inducers.Under light microscope, MGc80-3 cells, after treated with 1 mM dBcAMP, tended to be flat and disperse, and their volume gradually enlarged, with their uncleus relatively smaller and their shape rather regular. Morphological changes, like norma differentiated epithelial cells, were observed. The cells attached firmly, grew slowly, their growth curve showed inhibitory rate amounted to 52.87%, and cellular division exponent displayed their peak value 1.5 times less than that of MGc80-3 cells. It was clear that dBcAMP could effectively inhibit the multiplication activity of MGc80-3 cells. After dBcAMP treatment, remarkable changes of cell surface charges was indicated by cell electrophoresis, the ratio dropped to 3.043 from 3.988, and their re-tardant ratio reached up to 31.2%. cAMP content in cells after this treatment, detected by cAMP and cGMP radioimmunoassay, was enhanced by 2.42 times, and cAMP/cGMP ratio, by 1.73 times. Thus, cAMP level within MGc80-3 cells was raised obviously by dBcAMP. Heterotransplantation experiments showed that tuntorigenic rate of MGc80-5 cells (transplanted subcutaneously to BALB/c mice) amounted to 100%, and that of the cells after this treatment was only 5.6%. Their tumorigenic ability was extremely reduced.These results confirmed that dBcAMP was able to change malignant phenotypic characteristics of MGc80-3 cells and produce a reversed alteration: Thus, it has a remarkable inductive effect in differentiating gastric carcinoma cells. All these characteristics were also considered as the reference indexes in appraising reversed effect for the homologous cancer cells.

Effects of 4-(3-Chloro-Benzyl)-6,7-Dimethoxy-Quinazoline on Kinetics of P120-Catenin and Periplakin in Human Buccal Mucosa Squamous Carcinoma Cell Line

In order to detect molecular markers for the epidermal growth factor inhibitor 4-(3-chloro-benzyl)- 6,7-dimethoxy-quinazoline (tyrphostin), we investigated the kinetics of p120-catenin and periplakin in the human buccal mucosa squamous cancer cell line BICR 10 treated with 3 nM tyrphostin. Growth of BICR 10 cells was inhibited by treatment with tyrphostin. Although changes were not observed in the expression of EGFR and p120-catenin, expression of Akt, Src and periplakin in BICR 10 treated with 3 nM tyrphostin tended to decrease. In addition, phosphorylation of EGFR, Akt and Src was inhibited by treatment with tyrphostin. On immunocytochemical staining, immunoreactions with phosphorylated EGFR, phosphorylated Akt and phosphorylated p120-catenin were weak in BICR 10 treated with tyrphostin. There was a slight immunocy to chemical reaction to periplakin in BICR 10 cells induced by tyrphostin. In conclusion, the decrease in phosphorylation in EGFR and p120-catenin by tyrphostin, following the decrease in Src or Akt phosphorylation, may inhibit expression of several growth factors associated with the proliferation and migration of cancer cells.

Apoptosis Induced by Ginsenoside Rg3 in a Human Bladder Carcinoma Cell Line

OBJECTIVE This study was conducted to explore the effect of Rg3 on inhibition of proliferation and induction of apoptosis in bladder cancer cells. METHODS The EJ bladder cancer cell line was treated with Rg3 at various concentrations. Cell proliferation was measured by the MTT assay. Morphological changes in the cells were observed by fluorescent staining using Hoechst 33258. The cell cycle and apoptotic rate were analyzed by flow cytometry （FCM） and the expression of caspase-3 in cells was detected by immunocytochemistry. DNA ladder analysis was conducted by agarose gel electrophoresis. RESULTS Rg3 inhibited proliferation of EJ cells in a concentration-dependent manner, resulting in an IC50 for Rg3 at 48 h of 125.5 μg/ml. When treated with 150 μg/ml of Rg3 for 24 h and 48 h, the cells showed apoptotic morphological characteristics including condensed chromatin, nuclear fragmentation, apoptotic bodies and bright fluorescent granules as well as a higher caspase-3 expression. The FCM assay indicated that Rg3 altered the cell cycle and induced apoptosis of the EJ cells, when treated for 24 h and 48 h with 75 μg/ml of Rg3 as well as for 48 h with 150 μg/ml. The percentages of cells in the S phase and the GJM transition were increased, whereas the percentages of cells in the G0-G1 transition were decreased. The apoptotic rates were increased from （1.05±0.17）% in the control group cells to （8.41 ±0.98）%, （18.57±2.20）% and （33.98±1,64）% respectively. Significant changes in the DNA ladders, showed that the effects of Rg3 were displayed in a dose and time dependent manner. CONCLUSION The results suggest that Ginsenoside Rg3 exerts an inhibitory effect on proliferation of EJ cells by inducing apoptosis.

Role of the STAT3/survivin signaling pathway in the EML4-ALK-positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance

Objective This study investigated the role of the STAT3/survivin signaling pathway in the EML4-ALK- positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. The mecha- nism of resistance was studied. Methods Cell viability was determined using the MTT assay. Crizotinib-induced apoptosis in H2228 and H2228 crizotinib-resistant cells treated with the indicated doses of crizotinib was measured at different times （24 h, 48 h, 72 h） using flow cytometry. The levels of p-ALK, ALK, p-STAT3, STAT3, and survivin after treatment of cells with 0, 0.3, and 1 pM crizotinib for 72 h were determined using Western blot analysis. DNA sequencing was used to identify mutations in H2228 crizotinib-resistant cells. Results The crizotinib IC50 values in H2228 and H2228 crizotinib-resistant cells at 72 h were 334.5 nM and 3418 nM, respectively. The resistance index of 1-12228 crizotinib-resistant cells was 10.20. Crizotinib induced apoptosis in H2228 cells and reduced the levels of p-ALK, p-STAT3, and survivin. In contrast, no changes in the levels of p-ALK, p-STAT3, and survivin were observed in H2228 crizotinib-resistant cells. The mutations 2067G--,A and 2182G--,C in EML4-ALK were present in the H2228 crizotinib-resistant cells. Conclusion Crizotinib decreased the viability of H2228 cells in a dose- and time-dependent manner. In the STAT3/survivin pathway, downregulation of p-ALK, p-STAT3, and survivin might contribute to crizo- tinib-induced apoptosis in H2228 ceils. However, the STAT3/survivin pathway in H2228 crizotinib-resistant cells was unaffected by crizotinib treatment. Acquired resistance in H2228 cells might be related to ALK mutations.

Comparative Analysis of Protein Expression Concomitant with DNA Methyltransferase 3A Depletion in a Melanoma Cell Line

DNA methyltransferase 3A (Dnmt3a), a de novo methyltransferase, has attracted a great deal of attention for its important role played in tumorigenesis. We have previously demonstrated that melanoma is unable to grow in-vivo in conditions of Dnmt3a depletion in a mouse model. In this study, we cultured the Dnmt3a depletion B16 melanoma (Dnmt3a-D) cell line to conduct a comparative analysis of protein expression con-comitant with Dnmt3a depletion in a melanoma cell line. After two-dimensional separation, by gel electro-phoresis and liquid chromatography, combined with mass spectrometry analysis (1DE-LC-MS/MS), the re-sults demonstrated that 467 proteins were up-regulated and 535 proteins were down-regulated in the Dnmt3a-D cell line compared to the negative control (NC) cell line. The Genome Ontology (GO) and KEGG pathway were used to further analyze the altered proteins. KEGG pathway analysis indicated that the MAPK signaling pathway exhibited a greater alteration in proteins, an interesting finding due to the close relation-ship with tumorigenesis. The results strongly suggested that Dnmt3a potentially controls the process of tu-morigenesis through the regulation of the proteins (JNK1, p38α, ERK1, ERK2, and BRAF) involved in tu-mor-related pathways, such as the MAPK signaling pathway and melanoma pathway.

The establishment of a stable PC-3 cell line overexpressing micro RNA-145

Objective To establish stable prostate cancer bone metastasis cell line overexpressing microRAN-145 (miR-145)for the study of the mechanism of miR-145 in bone metastasis.Methods pMSCV-miR-145 plasmids and retroviruses of pMSCV-vector

柴胡皂苷D对人乳腺癌SK-BR-3细胞株增殖与凋亡的影响

目的:探讨柴胡皂苷D对人乳腺癌SK-BR-3细胞株增殖与凋亡的影响。方法:设置对照组、150 mg·L-1柴胡皂苷D组、100 mg·L-1柴胡皂苷D组、50 mg·L-1柴胡皂苷D组和25 mg·L-1柴胡皂苷D组,采用CCK-8实验方法检测柴胡皂苷D对人乳腺癌SK-BR-3细胞株增殖的影响;采用流式细胞术检测柴胡皂苷D对人乳腺癌细胞株SK-BR-3的细胞周期及细胞凋亡的影响。结果:柴胡皂苷D各浓度组细胞存活率分别为(85.3±4.13)%、(68.85±3.85)%、(45.28±3.25)%、(23.51±2.23)%,与对照组相比较,差异具有统计学意义(P<0.05)。柴胡皂苷D各浓度组G1期细胞比例分别为(58.23±2.21)%、(55.51±2.34)%、(45.67±3.23)%、(33.55±4.12)%,S期细胞的比例分别为(12.67±2.32)%、(10.34±2.45)%、(8.23±3.12)%、(7.34±3.98)%,G2期细胞所占的比例分别为(29.10±2.25)%、(34.15±3.45)%、(46.10±3.01)%、(59.11±2.34)%。柴胡皂苷D各浓度组G2期细胞所占比例与对照组比较,差异具有统计学意义(P<0.05)。柴胡皂苷D各浓度组细胞凋亡率分别为(9.00±1.35)%、(20.20±1.45)%、(38.50±2.50)%、(50.25±3.75)%,均高于对照组,差异具有统计学意义(P<0.05)。结论:柴胡皂苷D可明显阻滞SK-BR-3细胞株的增殖,将SK-BR-3细胞株的细胞周期阻滞于G2期,以促进该细胞的凋亡。

复方苦参注射液对PC-3细胞凋亡及cyclinE蛋白表达的影响

目的:研究复方苦参注射液对人前列腺癌PC-3细胞凋亡及cyclinE蛋白表达的影响。方法:用复方苦参注射液对体外培养的人前列腺癌PC-3细胞进行药物处理,采用MTT比色分析法测定复方苦参注射液不同剂量组对PC-3细胞生长的调控作用;采用流式细胞仪分析复方苦参注射液对PC-3细胞周期分布的影响,免疫细胞化学方法观察复方苦参注射液对PC-3细胞周期调控蛋白cyclinE的影响。结果:复方苦参注射液可抑制PC-3细胞的增殖,诱导其凋亡,改变细胞周期分布,使G0/G1期PC-3细胞比例增高,同时还可使cyclinE蛋白表达下降。上述作用具有时间和剂量的依赖性。结论:复方苦参注射液可诱导人前列腺癌PC-3细胞凋亡,改变细胞周期分布,影响细胞周期调控蛋白cyclinE的表达,从而抑制细胞增殖。

慢病毒介导下干涉酪氨酸激酶B表达对乳腺癌SK-BR-3细胞增殖的影响

目的 探讨慢病毒介导下干涉酪氨酸激酶B（TrkB）表达对乳腺癌细胞SK-BR-3增殖的影响及其作用机制.方法 对MCF-7、BCAP-37、SK-BR-3和MDA-MB231乳腺癌细胞进行筛选,选取TrkB表达水平相对较高的细胞进行慢病毒感染以干涉TrkB表达,构建稳定干涉TrkB的乳腺癌细胞系Lv-sh-TrkB,通过蛋白质印迹（Western blot）实验检测TrkB干涉效果,同时构建乱序干涉细胞系Lv-scramble.采用噻唑蓝方法、流式细胞仪检测干涉TrkB后细胞的增殖情况以及细胞周期变化.结果 4株乳腺癌细胞中,SK-BR-3细胞TrkB表达水平相对较高,通过慢病毒感染SK-BR-3细胞后筛选稳定干涉TrkB表达的细胞株Lv-sh-TrkB,Western blot实验结果表明,Lv-sh-TrkB细胞中TrkB表达水平明显低于空白对照和Lv-scramble 细胞.细胞增殖检测表明Lv-sh-TrkB细胞增殖速度明显低于空白对照和Lv-scramble细胞;流式细胞仪检测细胞周期结果显示Lv-sh-TrkB细胞发生G1期阻滞,空白对照、Lv-scramble和Lv-sh-TrkB细胞G1期比例分别为61％、62％和78％.结论 干涉TrkB基因后乳腺癌细胞SK-BR-3增殖受到明显抑制,细胞周期发生G1期阻滞.

奥曲肽对人类前列腺癌PC-3细胞凋亡及其对cyclinE蛋白表达的影响

目的:研究生长抑素类似物奥曲肽对人类前列腺癌PC-3细胞凋亡及其对cyclinE蛋白表达的影响。方法:用奥曲肽对体外培养的人类前列腺癌PC-3细胞进行药物处理,采用MTT比色分析法测定奥曲肽不同剂量组对前列腺癌PC-3细胞生长的调控作用;采用流式细胞术分析奥曲肽对PC-3细胞的周期分布的影响,免疫细胞化学方法观察奥曲肽对前列腺癌PC-3细胞周期调控蛋白cyclinE的影响。结果:奥曲肽可抑制前列腺癌PC-3细胞的增殖,诱导其凋亡,改变细胞周期分布,使G0/G1期细胞比例增高,S期比例降低,同时还可使cyclinE蛋白表达下降。上述作用具有剂量和时间依赖性。结论:奥曲肽可诱导前列腺癌PC-3细胞凋亡,改变细胞周期分布,影响细胞周期调控蛋白表达,从而抑制细胞增殖。

抑制葡萄糖调节蛋白78表达对阿霉素诱导人乳腺癌SK-BR-3细胞凋亡的增敏作用

目的:探讨特异性下调葡萄糖调节蛋白78(GRP78)对阿霉素(adriamyc in,ADR)诱导人乳腺癌SK-BR-3细胞凋亡的影响,以期为乳腺癌化疗提供新的靶点。方法:培养人乳腺癌SK-BR-3细胞,用ADR(1 mg/L)处理人乳腺癌SK-BR-3细胞0、6、12、24、36 h,W estern b lot检测GRP78的表达;ADR处理SK-BR-3细胞48 h后,用溴化丙啶染色测定细胞凋亡率;用小分子干扰RNA siRNA预处理SK-BR-3细胞,再予ADR同上处理,检测细胞凋亡率,比较siRNA作用前后细胞凋亡率的变化。结果:ADR可诱导内质网应激,上调GRP78的表达,SK-BR-3细胞对ADR诱导的细胞凋亡率<30%;siRNA可明显阻断GRP78的表达,显著增加ADR诱导的细胞凋亡作用,凋亡率达到(62.30±0.88)%(P<0.01)。结论:抑制GRP78的表达可增强人乳腺癌SK-BR-3细胞对ADR经内质网应激途径诱导细胞凋亡的敏感性,促进肿瘤细胞的凋亡,增强其抗肿瘤作用。

PROM2通过β-catenin/HER2通路调控乳腺癌细胞SK-BR-3的迁移、侵袭、增殖和凋亡

乳腺癌是当今威胁女性健康最主要的恶性肿瘤之一。虽然当前在乳腺癌的诊治方面获得了一定的进展,但其发病率和死亡率仍然较高,寻找有效的乳腺癌预后标记和治疗靶点是当前研究的热点。本研究通过对高通量基因表达数据库(Gene Expression Omnibus,GEO)分析发现,Prominin 2(PROM2)在诱导、增强和增殖成瘤能力的3组MCF7乳腺癌细胞中的表达,高于3组对照组MCF7细胞。且在诱导具有侵袭性的MCF10A细胞中的表达,高于未处理非侵袭性MCF10A细胞。研究选择了人乳腺癌细胞系SK-BR-3,成功地将过表达或敲减PROM2质粒转染到细胞中,并检测PROM2对SK-BR-3细胞的迁移、侵袭和增殖和凋亡的作用。划痕结果显示,敲减PROM2的SKBR-3细胞愈合面积更小(P<0.01),过表达PROM2的SK-BR-3细胞愈合面积更大(P<0.01);Transwell的结果显示,敲减PROM2的SK-BR-3细胞侵袭数量更少(P<0.01),过表达PROM2的SK-BR-3细胞侵袭数量更多(P<0.01);流式细胞术的结果显示,敲减PROM2的SK-BR-3细胞增殖能力减弱且凋亡比率增高(2.50%vs.0.93%),过表达PROM2的SK-BR-3细胞增殖能力增强且凋亡比率降低(0.43%vs.0.72%)。然后,探讨PROM2作用的可能机制。通过基因表达谱交互分析(gene expression profiling interactive analysis,GEPIA),对肿瘤基因图谱数据库(The Cancer Genome Atlas,TCGA)中的乳腺癌数据分析发现,PROM2与β-联蛋白(β-catenin)、人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)之间表达正相关。通过定量实时聚合酶链反应、免疫荧光和蛋白质印迹也发现,PROM2促进了β-联蛋白和HER2的表达。证明PROM2可能通过激活β-catenin/HER2通路,促进乳腺癌细胞SK-BR-3的迁移、侵袭和增殖,并抑制其凋亡。

miRNA-155 Modulates the Malignant Biological Characteristics of NK/T-Cell Lymphoma Cells by Targeting FOXO3a Gene

This study investigated the effects of miRNA-155 on malignant biological characteristics of NK/T-cell lymphoma cell lines and the possible mechanism. The expression of miRNA-155 was detected in lymphoma cell lines from different sources （SNK-6, YTS, Jurkat and DOHH2） by real-time PCR. Lentiviral vectors （pLL3.7） that could overexpress or downexpress miRNA-155 were constructed. Recombinant lentiviral particles were prepared and purified, and their titers determined. The expression of miRNA-155 in the infected SNK-6 cells and the cell proliferation were detected by PCR and CCK-8, respectively. Flow cytometry was used to determine the apoptosis of infected SNK-6 cells. The target of miRNA155 was predicted from Targetscan website. The effect of miRNA155 on FOXO3a expression was examined by Western blotting. The results showed that among the human NK/T-cell lymphoma cell lines SNK-6, YTS, Jurkat and DOHH2, the expression of miRNA-155 was highest in SNK-6. The infection efficiency of the recombinant lentivirns in SNK-6 was more than 70% at multiplicity of infection （MOI） of 100. The expression of miRNA-155 was significantly increased in SNK-6 cells infected by lentivirus vectors with high expression of miRNA-155 （4 times higher than the control group）, and profoundly decreased in those infected with lentivirnses with low expression of miRNA-155. The proliferation of letivirns-infected SNK-6 cells was decreased as the expression of miRNA-155 reduced. The apoptosis rate was increased with the reduction in the expression of miRNA-155. FOXO3a was found to be a possible target of miRNA155, as suggested by Targetscan website. Western blotting showed that the expression of FOXO3a was significantly elevated in SNK-6 cells with miRNA-155 inhibition. It was concluded that reduction in miRNA-155 expression can inhibit the proliferation of SNK-6 lymphoma cells and promote their apoptosis, which may be associated with regulation of FOXO3a gene.