Recombinant soluble TRAIL induces apoptosis of cancer cells

TRAIL is a tumor necrosis factor family member that selectively induces apoptosis of cancer cells but not of normal cells. To develop TRAIL into a potential cancer drug, three different sizes of soluble TRAIL fragments, including sTRAIL(74—281), sTRAIL(95—281) and sTRAIL(101—281), were expressed in E. coli and purified to homogeneity. Apoptosis assays indicated that sTRAIL(95—281) and sTRAIL(101—281), but not sTRAIL(74—281), can potently induce apoptosis of various cancer cell lines in 6 h, suggesting that the N-terminal fragment of aa101 has inhibitory effect on TRAIL-induced apoptosis. Moreover, we found that some cancer cells were resistant to TRAIL and the resistant cells could be converted into sensitive cells by treatment with the protein synthesis inhibitor cycloheximide, suggesting that one or more short-lived proteins are responsible for cells’ resistance to TRAIL.     

XIAP在TRAIL抗性细胞HCT116 bax-/-中的作用机制研究

为了探索结直肠癌细胞HCT116抗TRAIL诱导凋亡的分子机制,本课题以其抗性细胞HCT116 bax~(-/-)为实验对象进行了研究.通过利用目前最热的基因定点编辑技术Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)9系统将HCT116bax~(-/-)的XIAP(X-linked inhibitor of apoptosis protein)基因彻底敲除后,用TRAIL处理,发现其恢复了对TRAIL的敏感,形态学发生了明显凋亡,而且western blot检测显示PARP蛋白发生了完全剪切.由此证明敲除XIAP基因能克服HCT116 bax~(-/-)对TRAIL的抗性.这些发现对肿瘤细胞抗TRAIL的分子机理研究以及肿瘤的个性化治疗有非常重要的意义.     

The molecular mechanism of different sensitivity of breast cancer cell lines to TRAIL

Although Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of various cancer cells, some caner cell lines are resistant to TRAIL-induced cell death. To investigate the molecular mechanisms underlying TRAIL-resistance, two human breast cancer cell lines, MCF-7 (resistant to TRAIL) and MDA-MB-231 (sensitive to TRAIL), were used as a model system to analyze the different sensitivities to TRAIL cytotoxicity. PKCδ inhibitor rottlerin, but not MEK and ERK1/2 inhibitor U0126 nor PI3K inhibitor LY294002, was shown to enhance TRAIL-induced apoptosis in MCF-7 cells significantly, suggesting that PKCδ might play an important role in the resistance of MCF-7 cells to TRAIL. In contrast, rottlerin, U0126, and Ly294002 had no effect on MDA-MB-231 apoptosis induced by TRAIL under the same conditions. Further experiment showed that the combination of rottlerin and TRAIL cleaved PARP in the MCF-7 cells synergistically, but not in the MDA-MB-231 cells. The role of PKCδ in TRAIL-resistant MCF-7 cells was confirmed by knocking down the endogenous PKCδ expression using RNAi technology. Furthermore, caspase-3 reconstitution in MCF-7 cells was unable to alter PKCδ expression, suggesting that innate caspase-3 deficient in the cells does not cause PKCδ high expression. These data provide evidence for the first time that PKCδ plays a critical role in breast cancer cell lines to TRAIL cytotoxicity.     

Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

The use of plants as bioreactors for the expression oftherapeutic proteins has developed into a new field in biotechnology research[1]. Plant systems provide simple genetic manipulation, low production costs, and low risk of contamination by animal viruse…     

Human Soluble TRAIL Protein Inducing Apoptosis in Osteosarcoma Cell

This study is to examine the effect of human recombinant soluble TRAIL （TNF-related apoptosis-inducing ligand） protein inducing apoptosis in MG-63 human osteosarcoma cells. The inhibitive rates of TRAIL to MG-63 cells were detected by MTT assay. The apoptosis induced by TRAIL in MG-63 human osteosarcoma cells was analyzed with FACS and TUNEL and the apoptotic bodies were observed by transmission electron microscope. MTT assay showed that the inhibitive rates of 500, 1 000, 2 000 and 4 000 ng/mL TRAIL for 24 h were 10.1%, 24.3%, 50.6% and 97.7% respectively. Flow cytometric analysis showed that after MG-63 cells were treated with 2 gg/mL TRAIL for 6 h, obvious apoptotic peak would immediately appear before diploid peak. Human soluble TRAIL protein can quickly kill MG-63 osteosarcoma cells selectively, and may have potential value for clinical treatment of osteosarcoma.     

Effect of Quercetin on Breeding and Apoptosis of Cervical Cancer HeLa Cell and on Growth of Transplanted Tumor in Nude Mice

Effect of quercetin on HeLa cell system of cervical cancer was studied by methods of MTT and Annexin V-FITC/PI. The results show that quercetin has functions of inhibiting breeding of HeLa cells and inducing apoptosis of the cells. The total apoptosis rate is positively proportional to reaction duration and concentration of quercetin used. The maximum apoptosis rate being （88.76±2.35）% was obtained when the concentration was 50.0 μmol/L and the cells were treated with quercetin for 72 hours. Based on establishing a model of tumor of cervical cancer transplanted into nude mice, quercetin of different concentrations was injected into abdominal cavity of nude mice and situation of tumor growth was reviewed. The result showed that with quercetin concent＇ration increasing from 0 to 100.0 μmol/L, the transplantation volume and weight of the tumors decreased from （279.59±70.58） mm^3 and （0.145±0.019） g to （128.72±36.12） mm^3 and （0.089± 0.019） g respectively, while apoptosis rate of the transplanted tumor increased from （9.63±1.85）% to （34,98±0.47）%, which proved that quercetin inhibited increment of volume and weight of transplanted tumor in nude mice bodies.     

Construction and application of L929 cell model expressing human bcl-2 protein

Recombinant eucaryotic expression vector pLXSN/s-bcl-2 has been constructed by cloning human bcl-2 cDNA containing the full-length open reading frame into the vector pLXSN in sense orientation, and a mammalian cell model expressing human bcl-2 protein has been established by electroporating the recombinant vector into mouse L929 cells. bcl-2 expression in L929 cells has no effect on the cell growth and survival under normal culture conditions, but it can enhance the survival of the cell in the challenge of some apoptosis-inducing stimuli, including tumor necrosis factor α(TNF α) and staurosporine (STS).     

Anti-CD44 mAb remodels biological behaviors of spheroid cells with stemness from human ovarian cancer cell line SKOV-3

There is accumulating evidence that cancer stem cells (CSCs) play an important role in tumor progression. Novel strategies targeting CSCs have been widely researched. In the present study, we explored whether such CSCs existed in human ovarian cancer (OVCA) cell line and whether anti-CD44 antibody had effects on such subpopulation. We isolated and identified spheroid cells from SKOV-3. Then we used A3D8, an anti-CD44 mAb to treat spheroid cells with so-called "stemness". Effects of A3D8 on spheroid cells’ biological behaviors were examined. Our findings showed that there was a small subpopulation that had so-called "stemness" in SKOV-3 cell line. Against spheroid cells, A3D8 can (1) inhibit cell proliferation; (2) change cell cycle distribution and expression of p21, CDK2 and cyclinA; (3) enhance cisplatin (DDP)-induced apoptosis; (4) promote cell differentiation; (5) inhibit clone formation efficiency; (6) reduce invasive efficacy; (7) inhibit tumorigenicity. Thus, to sum up points which we have just showed, spheroid cells isolated from SKOV-3 can be used as an appropriate in vitro model for relevant study of human ovarian CSCs. And our results reasoned that anti-CD44 therapy may become a potential promising strategy for OVCA treatment.     

重组质粒pEgr-1-TRAIL辐射诱导乳腺癌细胞效应的实验研究

目的将具有辐射诱导表达特性的重组质粒p Egr-1-TRAIL转入乳腺癌MCF-7细胞中,诱导肿瘤杀伤基因TRAIL的表达,实现辐射和基因对MCF-7细胞的双重杀伤效应.方法用ELISA法检测不同剂量X射线诱导TRAIL表达的量效关系及2 Gy X射线照射后不同时间TRAIL的表达;用流式细胞仪检测不同处理组MCF-7细胞早期凋亡情况.结果不同剂量X射线照射转染p Egr-1-TRAIL重组质粒的乳腺癌细胞MCF-7,TRAIL表达量明显高于假照组(P0.001~0.05),其中5 Gy X射线照后表达量最高,TRAIL表达量为假照组的5.1倍.2 Gy X射线照射后4 h,TRAIL表达量明显升高(P0.05),并随时间延长逐渐增加,照射后32 h达峰值,表达量为照射前的4.6倍.MCF-7-p Egr-1-TRAIL/0 Gy组早期凋亡细胞百分数较MCF-7/0 Gy和MCF-7-p3.1Egr/0 Gy组明显增加(P0.01),随着照射剂量的增加,MCF-7-p Egr-1-TRAIL/5 Gy组细胞早期凋亡率与其他处理组比较均存在统计学意义(P0.001~0.05),MCF-7/0 Gy组细胞生长最快,而MCF-7-p Egr-1-TRAIL/8 Gy组细胞生长最慢.结论乳腺癌细胞转染p Egr-1-TRAIL重组表达质粒联合X射线照射能够增加MCF-7细胞凋亡,抑制其生长.     

Inhibiting effects ofS. acus Linnaeus extracts on GLT-82 tumor cell lines

HailongS. acus Linnaeus was chosen as the experimental material in the experiments and changes of cell morphology, forming rate of clone and changes of division index were conducted to identify the inhibiting effects of Hailong extract on human tumor cell lines (GLT-82). Four days after medication, most of the cells changed their normal morphology of tumor cells and became round, broken and even broke into pieces. The inhibiting rates could reach 75.1% on the fourth day. The division index reduced strongly and the clone could not form or the forming rate was very low. With the extract concentration increasing and the time prolonging, the inhibiting effect increased. These results indicate that Hailong has striking anti-tumor effects and will have a perfect future in the fields of treatment and prevention of cancer. Foundation item: Supported by the Science Foundation of the Education Department of Heibei Province (No. 2001263) Biography: LI Chun-xiang (1968-), female, Associate professor, research direction: biochemistry and molecular biology.     

Comparison of infection of different cell lines by uropathogenic Escherichia coli

Studying the interaction between uropathogenic Escherichia coil （UPEC） and uroepithelial cells is important in elucidating the pathogenesis of urinary tract infection. In this study, the African green monkey kidney cells （Vero）, human kidney carcinoma cells （Ketr-3） and bladder carcinoma cells （EJ） were infected by UPEC132, a clinical strain isolated from Tianjin, China, and were compared for their capacities to allow the adherence and invasion by this strain. The results revealed that all these cell lines could be attached and invaded by UPEC132. The adherence rates for Vero, Ketr-3 and EJ cells were （49,20 ±7.55）%, （55.22 ±4.09）% and （73.20 ±5.26）%, respectively, and invasion frequencies were （2.61 ±0.32）×10^-3, （3.00 ±0.34）×10^-3 and （3.25 ± 0.20）×10^-3, respectively. The statistical analysis showed that the adherence rate for EJ cells was significantly higher than those for the other two cell lines （P〈0.05）, and the invasion frequencies for EJ and Ketr-3 cells had no statistical differences （P〉0.05） but were higher than that for Vero cells （P〈0.05）. Three cell lines were detected for the receptors for P pill of UPEC by using indirect immunofluorescence. The results showed that receptors existed on the surfaces of all cell lines, and the highest distribution was found on the surface of EJ cells. Additionally, the invasion of EJ cells by recombinant UPEC132/pSELECT-GFP could be directly visualized using confocal microscopy. These data strongly implicated that EJ cells could be more easily infected by UPEC132 than the other cells, and thus could serve as a good experimental target for further investigation of UPEC infection.     

人参皂甙Rh2抗肿瘤作用机制的研究

恶性肿瘤治疗至今尚无好的方法.传统化疗药因其缺乏特异性的细胞毒性作用,治疗中容易造成正常细胞的损伤,从而引起一系列的并发症.人参皂甙Rh2是人参中提取的天然活性成分,具有很高的抗肿瘤活性,而对正常细胞无毒副作用.文章从人参皂甙Rh2的抗肿瘤作用机制(抑制细胞增殖、诱导细胞凋亡、诱导分化、抑制肿瘤DNA合成、提高荷瘤机体的免疫功能和抑制肿瘤转移、抗肿瘤耐药性增强、其他化疗药物对耐药肿瘤作用)等方面进行研究.     

The apoptosis and cell cycle changes of SUD4 and DOHH2 induced by topotecan

Topotecan (TPT), a semisynthetic analogue of the natural product camptothecin is a cell cycle-specific drug with antitumor activity. To clarify the effect of TPT on SUD4 and DOHH2 cell line in this study, we examined the apoptosis and cell cycle changes of the two human cancer cell lines by exposing to TPT for 18 hours at various concentrations. The linear relationship between apoptosis cell number and the concentration of TPT was observed by means of Flow Cytometry and Annexin V assay. Then, DOHH2 cell is much more sensitive to TPT than SUD4 cell. In addition, Cell Question Software Assay showed positive relationship between the frequency of cells accumulated in S-phase and the concentration of TPT. The least concentration of TPT to change cell cycle is 5 nmol·L⁻¹ in both cell lines. These results suggest that the inducing apoptosis of cancer cells is one of mechanism of TPT antitumor activity. Feng Xiaorong: born in 1965, Post-doctoral Research Fellow, Engaging in Cell and Molecular Biology.     

Cloning of murine BRI3 gene and study on its function for inducing cell death

To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3) treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bcl-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFa mediated cytotoxicity.     

Quantitative detection of ING1 mRNA under different gene regulation based on molecular beacon

As an important tumor suppressor, normal expres- sion of ING1 is associated with the maintenance of cell normal physiological functions. Its low expression of- ten causes aberrant events of cell survive, growth and differentiation[1] which results in loss…     

Apoptosis of Spodoptera litura larval hemocytes induced by heavy metal zinc

By adding different amount of zinc into the artificial medium of the insect larvae, the zinc-induced apoptosis of the larvae haemocytes of the herbivorous insect Spodoptera litura Fabricius was investigated with flow cytometer. The results showed that the increase of zinc dose in the artificial feed led to the accumulations of zinc in the larval hemolymph and fat body, and more zinc was accumulated in fat body than in hemolymph. The apoptosis of hemocytes was significantly induced at high zinc concentration （1000 mg·kg^-1） in the insect diet, and the apoptosis rate was 63.63%, which was remarkably higher than that at control and lower concentrations （50--500 mg·kg^-1）. This suggests that the high dose of zinc in the artificial diet of S. litura larvae could induce the apoptosis of the larval hemocytes of S. litura.     

细菌内毒素诱导人肝癌细胞凋亡的实验研究

目的:探讨细菌内毒素对人肝癌细胞株7721的影响程度.方法:用含有细菌内毒素1000 EU/mL、500EU/mL、100 EU/mL、50 EU/mL、0EU/mL的DMEM细胞培养液来培养7721细胞,通过细胞计数来绘制细胞生长曲线,台盼蓝排除法测定细胞活率、用DNA梯形条带法及TUNEL原位法检测细胞凋亡情况.结果:细菌内毒素对肝癌细胞7721生长有明显的抑制作用,且呈剂量依赖性.此种抑制是通过诱导细胞凋亡达到的,肿瘤坏死因子检测结果没有发现规律性.结论:内毒素在体外可以诱导肺腺癌细胞的凋亡,且存在明显的剂量关系,并且此凋亡不是依赖TNF-α途径发生的,为临床上诱导肿瘤细胞凋亡,从而为预防和治疗肿瘤提供了理论依据.