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.

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line

AIM： To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53（N 15）-antennapedia （Ant）, and study its effect on HepG2 cells. METHODS： Plasmid pLenti6/V5-NT4 p53（N15）-Ant was constructed incorporating the following functional regions, including signal peptide sequence and proregion of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma （HepG2） cells were used for transfection. 3-[4,5-climethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide （MI-I） assay, lactate dehydrogenase （LDH） release assay, transmission electron microscopy （TEM） and flow cytometric analysis （FCM） were employed to investigate the effects of LV-NT4（Si）- p53（N15）-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4（Si）-p53（N15）-Ant on tumor growth in nude mice.RESULTS： LV-NT4（Si）-p53（N15）-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4（Si）-p53（N15）-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4（Si）-p53（N15）-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4（Si）-p53（N15）-Ant （P 〈 0.01）. Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, but no significant changes in HepG2 cells infected with LV-EGFR Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4（Si）-p53（N15）-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4（Si）-p53（N15）-Ant treatment group （682 IU/L） than in control group （45 IU/L, P 〈 0.01）. The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4（Si）- p53（N15）-Ant could induce two different kinds of cell death： necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4（Si）-p53（N15）-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4（Si）-p53（N15）-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight. CONCLUSION： LV-NT4（Si）-p53（N15）-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.

2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮诱导Hep3B人肝癌细胞凋亡的机制

近年来,紫草萘醌类衍生物因其临床应用受到副作用的限制。为寻找副作用小疗效高的新型抗肿瘤药物,合成了2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮,并对其化学结构进行了鉴定。同时研究了2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮对人肝癌细胞活力、凋亡的影响及其潜在机制。研究结果表明,通过MTT检测其细胞活力,发现2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮显著降低了人肝癌细胞系的细胞活力。蛋白免疫印迹结果表明,该化合物可通过上调Cle-caspase3、Bad和Bax凋亡相关蛋白表达水平来诱导肝癌细胞Hep3B凋亡。为进一步检测2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮诱导Hep3B细胞发生凋亡的原因,使用荧光探针JC-1检测了2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮处理后Hep3B细胞内线粒体膜电位的变化。结果发现,与对照组相比,药物处理组线粒体膜电位显著下降,绿色荧光增强,红色荧光减弱,说明2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮能通过线粒体损伤来诱导Hep3B细胞凋亡。由于2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮显著诱导Hep3B细胞凋亡。因此,2-(4-甲氧基-苯巯基)-5,8-二甲氧基萘-1,4-二酮具有良好的抗肿瘤活性。

Mutations and altered expression of p16^(INK4a) in human pancreatic carcinoma cell lines with different potential of metastasis

Objective:To analyze the p16INK4a genomic alteration and expression status in 3 human pancreatic carcinoma cell lines with different potential of metastasis. Methods:Using PCR-SSCP, Dot-blot and immunohistochemistry, the p16INK4a genomic mutation and expression were analyzed on DNA, mRNA and protein levels in 3 human pancreatic carcinoma cell lines Patu8902, Patu8988 and SW1990, which had different potential of metastasis. Results: (1) On DNA level: there was no deletion of p16INK4a Exon Ⅰ in 3cell lines; p16INK4a Exon Ⅱ was only deleted in Patu8902 while no deletion in Patu8988 and SW1990. No insertion, microdeletion and point mutation were found in the 3 cell lines. (2) On RNA level: the expression of p16INK4a protein was negative in Patu8902, low expressed in SW1990, but highly expressed in Patu8988.(3) On protein level: P16 protein was strongly stained in Patu8988, much lower in SW1990, but not stained in Patu8902. Conclusion:The genomic type and expression of p16INK4a are quite different in 3 pancreatic carcinoma cell lines which have different potential of metastasis. It is suggested that genomic homozygous deletion and low expression of mRNA might relate to the potential of metastasis of pancreatic cell lines. In other words, dysfunction of p16INK4a might be an important mechanism in the metastasis of pancreatic carcinoma.

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.

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.

In Vivo Animal Model Evaluation of a Powerful Oral Nanomedicine for Treating Breast Cancer in BALB/c Mice Using 4T1 Cell Lines without Chemotherapy

Nanopharmaceuticals containing quantum dot nanoparticles (Q-Dot NPs) for treating serious cancers such as breast cancer have made fantastic proposals. In this study, ZnO quantum dot NPs are formulated via ZnO@PVP nanopolymer as co-assistants coordinating with efficacious suitable wetting agents, PEG-binding compound, and W/O emulsifier for producing eco-friendly water-based nanodrug. Several characterization techniques containing SEM, TEM, FTIR, photoluminescence, zeta potential, and UV-Vis absorption were employed for ZnO Q-Dot NPs in nanodrug. This work aims to investigate the anti-tumor effects of such nanomedicine on the 4T1 breast cancer cell line in BALB/c mice, being elaborated through intraperitoneal, injection (IVP) and oral therapy. The impressive findings showed that ZnO nanodrug caused changes in blood factors, having the most effectiveness at 40 μg/ml concentration after two weeks of oral treatments. The significant increase in white blood cells (WBC) neutrophils and meaningful decreases in lymphocytes and especially cholesterol were powerful simultaneous impacts, successfully treating malignant breast cancer masses. In this significant animal model research for breast cancer, the sick mice recovered entirely and even had a safe space to mate. Histopathological results showed no evidence of breast tumor formation or metastasis in the group treated with nanodrug and their children. This nanomedicine has a therapeutic effect, and is ready to be applied for treating volunteer breast cancer patients. However, its prevention (inhibitory) effect can also be analyzed and added to current data in future studies.

Construction of Cox7a2 fluorescent vector and its effect on cytochrome C oxidase activity in mouse Sertoli cell line TM4

Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was

雄黄诱导人弥漫性大B淋巴瘤SU-DHL-4细胞凋亡及其机制的初步研究

本研究旨在探讨雄黄(Realgar,As4S4)诱导人弥漫性大B淋巴瘤SU-DHL-4细胞凋亡及其可能的机制。采用MTT法观测雄黄对SU-DHL-4细胞的增殖抑制作用,应用流式细胞术检测药物对SU-DHL-4细胞周期的影响及其诱导凋亡情况,利用琼脂糖凝胶电泳方法观察凋亡细胞的DNA降解情况,通过透射电子显微镜技术观察药物作用前后细胞超微结构的变化,采用Western blot方法检测药物诱导SU-DHL-4细胞48 h后Caspase-3、Bcl-2以及Bax蛋白表达情况。结果表明:20、40、80μmol/L的雄黄均可抑制SU-DHL-4细胞增殖,抑制率呈时间-剂量依赖性(r=0.982;P<0.05);AnnexinV/PI双染流式细胞术检测细胞凋亡率显示雄黄作用SU-DHL-4细胞48 h后,随着药物浓度的增加,早期凋亡细胞所占比率增高(P<0.05);PI单染流式细胞术检测细胞周期发现,雄黄作用SU-DHL-4细胞48 h后处于细胞周期S期的细胞显著减少(P<0.05);透射电子显微镜下观察雄黄作用SU-DHL-4细胞48 h后各浓度组细胞,均呈典型凋亡的改变,偶见坏死细胞;琼脂糖凝胶电泳结果显示,各浓度实验组雄黄干预SU-DHL-4细胞48 h后,细胞核DNA降解呈梯状;Western blot结果显示,雄黄作用SU-DHL-4细胞48 h后,Bcl-2蛋白表达降低,Bax和Caspase-3蛋白表达增加。结论:雄黄可以诱导人弥漫性大B淋巴瘤细胞凋亡,其作用机制可能是通过抑制细胞增殖、阻滞细胞周期、上调Caspase-3和Bax蛋白,下调Bcl-2蛋白而诱导细胞凋亡。

表观遗传学药物对弥漫大B细胞淋巴瘤SU-DHL-4细胞株增殖及SHP-1基因表达的影响

目的探讨去甲基化药物5-氮杂-2’-脱氧胞苷(5-aza-2'-deoxycytidine,5-Aza-CdR)及组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸(N-Hydroxy-N-phenyloctanediamide,SAHA)对弥漫大B细胞淋巴瘤SU-DHL-4细胞株增殖及抑癌基因蛋白酪氨酸磷酸酶-1基因(Src-homologydomain2-containingprotein tyrosine phosphatase-1,SHP-1)表达的影响。方法四甲基偶氮唑蓝(MTT)法检测5-Aza-CdR及SAHA对SU-DHL-4细胞株增殖的影响,SYBR Green相对定量反转录聚合酶链反应(RT-PCR)方法检测SHP-1基因表达水平。结果5-Aza-CdR及SAHA均可抑制SU-DHL-4细胞的增殖,且表现为浓度依赖性;1μmol/L5-Aza-CdR及2μmol/SAHA联合应用后,SU-DHL-4细胞的增殖抑制率增高为52.23%,且不同药物浓度间吸光度比较(P<0.01);5-Aza-CdR及SAHA均能使SU-DHL-4细胞中SHP-1重新表达,且联合应用后表达量更高。结论表观遗传学药物能显著抑制弥漫大B细胞淋巴瘤SU-DHL-4细胞株增殖,可能与其诱导的SHP-1基因的表观遗传改变和SHP-1的再表达有关。

血清4型禽腺病毒penton蛋白单克隆抗体的制备及鉴定

【目的】制备和鉴定血清4型禽腺病毒(FAdV-4)五邻体(penton)蛋白单克隆抗体,为深入探究penton蛋白在FAdV-4感染致病机制中的作用及为抗原检测试剂研发提供抗体材料。【方法】以原核表达的FAdV-4 penton重组蛋白免疫6~8周雌性BALB/c小白鼠,加强免疫3次后,取鼠脾脏细胞与骨髓瘤细胞SP2/0进行细胞融合,通过penton-酶联免疫吸附剂测定(ELISA)和FAdV4 virus-ELISA方法分别筛选阳性杂交瘤细胞株,对筛选出的单克隆抗体杂交瘤细胞株进行亚类鉴定、特异性和广谱性鉴定及间接免疫荧光和稳定性试验。【结果】亚类鉴定结果表明,筛选获得7株能稳定分泌抗FAdV-4 penton蛋白的杂交瘤细胞株(1D11、2B3、2C4、2C9、6B3、8F12和8G11),其轻链均为Kappa链,重链3株为IgG1,4株为IgG2b;特异性检测结果表明,7株单克隆抗体只与FAdV-4反应,不与其他11种血清型FAdV及NDV、ARV、EDSV和IBV等常见禽病病原体发生交叉反应;广谱性鉴定结果表明,7株单克隆抗体均可与19株FAdV-4分离株结合,广谱性好;间接免疫荧光试验结果表明,7株单克隆抗体均与感染FAdV-4的鸡胚肝细胞(CEL)结合,出现明亮的绿色荧光。对筛选出的3株单克隆抗体(6B3、8F12和8G11)进行稳定性检测,发现其稳定性良好。【结论】通过单克隆抗体制备和鉴定获得3株与FAdV-4蛋白结合效果较好并具有特异性和广谱性的FAdV-4 penton蛋白杂交瘤细胞株(6B3、8F12和8G11),能稳定分泌penton蛋白单克隆抗体,为深入研究penton蛋白在FAdV-4感染致病机制中的作用提供单抗,为FAdV-4抗原检测试剂的研发打下基础。

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.

Implanting 1.1B4 human β-cell pseudoislets improves glycaemic control in diabetic severe combined immune deficient mice

AIM To investigate the potential of implanting pseudoislets formed from human insulin-releasing β-cell lines as an alternative to islet transplantation. METHODS In this study, the anti-diabetic potential of novel human insulin releasing 1.1B4 β-cells was evaluated by implanting the cells, either as free cell suspensions, or as three-dimensional pseudoislets, into the subscapular region of severe combined immune deficient mice rendered diabetic by single high-dose administration of streptozotocin. Metabolic parameters including food and fluid intake, bodyweight and blood glucose were monitored throughout the study. At the end of the study animals were given an intraperitoneal glucosetolerance test. Animals were then culled and blood and tissues were collected for analysis. Insulin and glucagon contents of plasma and tissues were measured by insulin radioimmunoassay and chemiluminescent enzyme-linked immunosorbance assay respectively. Histological analyses of pancreatic islets were carried out by quantitative fluorescence immunohistochemistry staining. RESULTS Both pseudoislet and cell suspension implants yielded well vascularised β-cell masses of similar insulin content. This was associated with progressive amelioration of hyperphagia(P < 0.05), polydipsia(P < 0.05), body weight loss(P < 0.05), hypoinsulinaemia(P < 0.05), hyperglycaemia(P < 0.05- P < 0.001) and glucose tolerance(P < 0.01). Islet morphology was also significantly improved in both groups of transplanted mice, with increased β-cell(P < 0.05- P < 0.001) and decreased alpha cell(P < 0.05- P < 0.001) areas. Whereas mice receiving 1.1B4 cell suspensions eventually exhibited hypoglycaemic complications, pseudoislet recipients displayed a more gradual amelioration of diabetes, and achieved stable blood glucose control similar to non-diabetic mice at the end of the study. CONCLUSION Although further work is needed to address safety issues, these results provide proof of concept for possible therapeutic applicability of human β-cell line pseudoislets in diabetes.

Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells： an insight into the efflux-metabolism alliance

Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients （Papp） of （ - ） and （ ＋ ）CLA were higher in the ab- sorptive direction than those in the secretory direction with efflux ratios（ER） of 0. 709 ± 0.411 and 0. 867± 0. 250 （ Х10^-6 -1 cm · s ）, respectively. Their bidirectional transports were significantly reduced by （75.6 ± 87.5）% af- ter treatment with verapamil （ a P-glycoprotein inhibitor） that increased the rate of metabolism by CYP3 A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of （ - ） and （ ＋ ） CLA with ERs being 2. 934 ± 1. 432 and 1. 877 ± 0. 148 （ Х 10^-6 cm/s） respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enanti- omers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following （ - ） or （ ＋ ）-isomer treatment alone. Furthermore, the uptake of （ - ）CLA was more than that of （ ＋ ）CLA in the transfected cells. Incubation with ketoeonazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexametha- sone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharma- cology of （？） CLA as a candidate drug for treatment of Alzheimer＇ s disease.

Effect of realgar on telomerase activity and hTERT-mRNA expression in NB4 cells

Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expression of hTERT-mRNA was analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Flow cytometry using PI staining was applied to analyze the cell cycle and apoptosis. Results: Treatment of NB4 cells with 155, 300, 600 μg/L realgar reduced telomerase activity significantly accompanying with decrease of hTERT-mRNA and increasing cell apoptosis. G2/M phase arrest appeared when treated with realgar in 300, 600 μg/L. Conclusion: It is suggested that telomerase activity of NB4 cells can be specifically inhibited by realgar through the down-regulation of hTERT gene expression. G2/M phase arrest and apoptosis by realgar in NB4 cells might be related to the reduction of telomerase activity and hTERT-mRNA expression.

Direct interaction between Rab5a and Rab4a enhanced epidermal growth factor-stimulated proliferation of gastric cancer cells

BACKGROUND Gastric cancer(GC)is one of the leading causes of cancer-related death worldwide.Although targeted therapies such as antibodies against human epidermal growth factor receptor 2 or vascular endothelial growth factor receptor 2 have been widely used in the treatment of metastatic cancer,the overall outcomes are poor.Therefore,elucidation of the mechanism underlying cancer progression is important to improve prognosis.Overexpression of the Rab5a gene has been confirmed to correlate with tumorigenesis of many cancers,but the mechanism underling,especially of GC,is still unclear.AIM To investigate the effects of Rab5a overexpression on the tumorigenesis of GC.METHODS First,the expression levels of Rab5a and Rab4a in primary tumorous tissues of GC patients diagnosed between 2015 and 2018 were analyzed.Then we constructed HGC-27 cell lines overexpressing green fluorescent protein-Rab5a or red fluorescent protein-Rab4a and investigated the interaction between Rab5a or Rab4a using Western blotting,co-immunoprecipitation,confocal microscopy,and colocalization analysis.Finally,epidermal growth factor-stimulated proliferation of these cell lines was analyzed using cell counting kit-8 cell viability assay.RESULTS Compared with normal gastric tissues,the expression levels of Rab5a and Rab4a increased progressively both in paracancerous tissues and in advanced cancerous tissues.Epidermal growth factor could promote the proliferation of HGC-27 cells,especially Rab5a-overexpressing HGC-27 cells.Notably,Rab5a and Rab4a cooverexpression promoted the proliferation of HGC-27 cells to the greatest extent.Further analysis identified a direct interaction between Rab5a and Rab4a in HGC-27 cells.CONCLUSION Co-overexpression of Rab5a and Rab4a in GC may promote the endosomal recycling of epidermal growth factor receptor,which in turn contributes to poor prognosis and tumor progression in GC patients.Inhibition of Rab5a or Rab4a expression might be a promising therapy for refractory GC.

Intrastriatal glial cell line-derived neurotrophic factors for protecting dopaminergic neurons in the substantia nigra of mice with Parkinson disease

BACKGROUND： Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor （GDNF） injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE： To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease （PD）, and analyze the action pathway. DESIGN： A controlled observation. SETTING： Neurobiological Laboratory of Xuzhou Medical College. MATERIALS： Twenty-four male Kunming mice of 7 - 8 weeks old were used. GDNF, 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine （MPTP） were purchased from Sigma Company （USA）; LEICAQWin image processing and analytical system. METHODS： The experiments were carded out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group： GDNF 4-day group, phosphate buffer solution （PSB） 4-day group, GDNF 6-day group and PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 μ L GDNF solution （20 μ g/L, dispensed with 0.01 mol/L PBS） injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. ② On the 12^th day after model establishment, the midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase （TH） and calcium binding protein （CB） with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES： Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS： All the 24 mice were involved in the analysis of results. The numbers of TH^＋ and CB^＋ neurons in the GDNF 4-day group （54.33±6.92, 46.33±5.54） were obviously more than those in the PBS 4-day group （27.67±5.01, 21.50±5.96, P 〈 0.01）. The numbers of TH^＋ and CB^＋ neurons in the GDNF 6-day group （75.67±5.39, 69.67±8.69） were obviously more than those in the PBS 6-day group （27.17±4.50, 21.33 ±5.72, P 〈 0.01） and those in the GDNF 4-day group （P 〈 0.01 ）. CONCLUSION： Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression.

EFFECTS OF PML AND PML/RMRα ANTISENCE OLIGO-NUCLEOTIDE ON PROMYELOCYTIC LEUKEMIA CELL NB4

Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RAR( (promyelocytic leukemia/retionic acid receptor() antisense oligonucleotides on cell growth, expression of PML-RAR( mRNA and PML-RAR(/PML protein location of NB4 cell lines. Methods: RT-PCR was used for detecting PML-RAR( mRNA expression, trypan blue exclusion for cell count, methylcellose assay for leukemic colony forming unit detection, immuno- fluorescence for PML-RAR(/PML protein location. Results: Both anti-PML start codon region antisence (STAS) and anti-PML-RAR( fusion region antisence (FUAS) could inhibit cell growth and the formation of acute myelocytic colony forming unit of cells(AML-CFU). Cells become partial differentiated at days 5, being more obvious in FUAS-treated cells than in STAS ones. Down regulation of PML-RAR( mRNA expression occurred at 24 hours in STAS and FUAS-treated cells and maintained for up to 72 hours. Immuno-fluorescence analysis with anti-PML monoclonal antibody showed a remarkable decrease even complete disappearance of microgranules. The residual granules became enlarged as discrete dots (<10 per cell), similar to normal POD structure in some STAS-treated cells at 24 hours. At 72 hours, nearly all the granules disappeared. Similar changes were observed in FUAS-treated cells. Conclusion: Both PML and PML-RAR( antisence oligonucleotides can specially block the expression of PML-RAR( at mRNA and protein levels. PML protein is implicated in the regulations of cell differentiation.

Influence of Glyphaea brevis twig extract on nucleus, tight junctions and expression of inhibin-β, stem cell factor, and androgen binding protein in TM4 Sertoli cells

Objective: To examine the influence of Glyphaea (G.) brevis twig extract on the mitochondrial dehydrogenase activity, integrity of the tight junctions between adjacent cells, mitochondria, apoptosis, nucleus and expression of inhibin-β, stem cell factor, and androgen binding protein in TM4 Sertoli cells. Methods: TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells. TM4 Sertoli cells were exposed to G. brevis twig extract (0.1, 1.0, 10.0, 100.0, or 1000.0μg/mL) for 24, 48 and 72 h. Parameters studied included cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay], mitochondrial membrane potential (tetra methyl rhodamine ethyl ester dye), transepithelial electrical resistance, apoptosis (Annexin V Alexa Fluor?488/propidium iodide assay) and mRNA expression (quantitative reverse transcription polymerase chain reaction). Results: G. brevis twig extract had no cytotoxic impact on cell viability, thus, considerably increasing the activity of mitochondrial dehydrogenase enzyme after 24 and 72 h exposure. Transepithelial electrical resistance values revealed substantial (P<0.05) rise in treated groups, especially after 72 h of treatment. Moreover, there was a significant decrease in mitochondrial depolarization of TM4 Sertoli cells exposed to G. brevis twig extract when compared to controls. In addition, G. brevis twig extract significantly reduced necrosis and apoptosis of TM4 Sertoli cells when compared to control. Nevertheless, fluorescence microscopy revealed that the nuclei were egg-shaped and marked uniformly with consistent cell shape at the middle of the TM4 Sertoli cells. Significant stimulatory effects were observed on mRNA levels of inhibin-β, androgen binding protein and stem cell factor. Conclusions: G. brevis twig extract may increase the secretory roles of TM4 Sertoli cells, cells proliferation, as well as cell-cell tight junction integrity. Thus, G. brevis twig may enhance spermatogenesis.

稳定表达结核分枝杆菌38抗原的EL-4细胞株的建立及鉴定

将结核分枝杆菌的38抗原基因片段经PCR方法扩增并插入到pcDNA3真核表达载体中,通过脂质体转染EL-4细胞,经G418筛选,用RT-PCR方法和ELISA方法检测整合和表达情况。结果成功地构建了pcDNA3-38抗原重组质粒,转染细胞中可检测到38抗原的存在,PCR扩增也证实38抗原基因已稳定整合于EL-4细胞的染色体中。本实验为今后在小鼠体内检测结核DNA疫苗激发的细胞毒性T淋巴细胞(CTL)反应奠定了基础。