菠萝蛋白酶对宫颈癌细胞HeLa增殖及凋亡的影响

目的:探讨菠萝蛋白酶对人宫颈癌细胞HeLa增殖和凋亡的影响。方法:将人宫颈癌HeLa细胞用不同剂量的菠萝蛋白酶处理48h后,光学显微镜下观察其形态学变化;采用MTT法检测不同剂量菠萝蛋白酶作用不同时间(24h、48h、72h)后对HeLa细胞增殖的影响;然后采用FITC-annexin V/PI双染色法检测不同剂量处理组HeLa细胞的凋亡情况。结果:菠萝蛋白酶对HeLa细胞的生长抑制作用呈明显的时间、剂量依赖性;且随着剂量增加,能够诱导HeLa细胞发生凋亡,与对照组相比具有显著的统计学意义(P<0.05)。结论:菠萝蛋白酶能明显抑制宫颈癌HeLa细胞的增殖,可能是通过诱导细胞凋亡的发生,从而发挥其抗肿瘤效应。

丙型肝炎病毒NS3基因真核表达载体的构建及表达

目的:构建丙型肝炎病毒(HCV)非结构基因NS3的真核表达载体,并分析其在HeLa细胞中的表达。方法:以HCV全长cDNA质粒为模板进行PCR扩增,获得全长NS3基因,将其插入克隆载体pMD18-T中,鉴定后与真核表达载体pcDNA3.1(+)重组,用脂质体介导法转染HeLa细胞,间接免疫荧光法及Western blot鉴定转染后HCVNS3蛋白的表达。结果:PCR扩增的NS3序列正确,构建的真核表达载体成功转染HeLa细胞,表达的NS3蛋白相对分子量为70kD。结论:成功构建了真核表达载体pcDNA3.1(+)/NS3,并在HeLa细胞中获得表达。

Influences of Melatonin on the Growth of HeLa Cells

Aim To investigate the influences of melatonin (MT) on the growth of HeLa cells in vitro. Methods Theantiprolfferation activities of MT were evaluated in HeLa cells by means of trypan blue dye exclusion and MTT vital staining.The morphological changes of HeLa cells induced by MT were observed under transmission electronic microscope. Cell divisioncycle influenced by MT was assessed by a flow cytometry. Results MT produced a certain inhibition of HeLa cells at the con-centration of 2 mmol@ L-1 and prolonged the TD. The fraction of cells inhibited was 61.0%. The IC. so of HeLa cells exposed toMT for 96 h was 2.039 mmol@ L- 1. The flow cytometric analyses showed that exposure to MT for 72 h reduced the number ofHeLa cells in phase S. Under electronic microscope, the HeLa cells exposed to MT for 72 h displayed morphological changesof necrosis, apoptosis, more hetero-chromosome and less somatic chromosome. Conclusion MT showed certain influences onthe growth of HeLa cells. Its mechanism may probably be attributable to reduction of the number of cells in phase S.

Dynamic distribution of TTK in HeLa cells: insights from an ultrastructural study

Entry into mitosis is driven by signaling cascades of mitotic kinases.Our recent studies show that TTK,a kinetochore-associated protein kinase,interacts with CENP-E,a mitotic kinesin located to corona fiber ofkinetochore.Using immunoelectron microscopy,here we show that TTK is present at the nuclear pore adjacent complex of interphase HeLa cells.Upon nuclear envelope fragmentation,TTK targets to the outermostregion of the developing kinetochores ofmonoorient chromosome as well as to spindle poles.After stable attachment,throughout chromosome congression,TTK is a constituent of the corona fibers,extending up to 90 nm away from the kinetochore outer plate.Upon metaphase alignment,TTK departs from the kinetochore and migrates toward the centrosomes.Taken together,this evidence strongly supports a model in which TTK functions in spindle checkpoint signaling cascades at both kinetochore and centrosome.

C/EBPα regulates SIRT1 expression during adipogenesis

SIRT1 plays an important role in adipogenesis, but how SIRT1 is regulated in adipogenesis is largely unknown. In this study, we show that both SIRT1 protein and mRNA levels were increased along with CCAAT/enhancer-binding protein a （C/EBPa） during adipocyte differentiation. C/EBPa, but not C/EBPap30, activated SIRT1 promoter in both HeLa cells and 3T3-L1 preadipocytes. Furthermore, C/EBPa upregulated SIRT1 mRNA and protein levels in HeLa cells and increased SIRT1 expression in a p53-independent manner in Soas2 cells. In preadipocytes, ectopic expression of C/EBPa upregulated SIRT1 protein level and knockdown of C/EBPa led to the decrease of SIRTI pro- tein level. Moreover, by promoter deletion analysis, gel shift assay and chromatin immunoprecipitation, we found that C/EBPa bound to the SIRT1 promoter at a consensus C/EBPα binding site. These data demonstrate that C/ EBPα regulates SIRT1 expression during adipogenesis by directly binding to the SIRT1 promoter.

MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION

To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher study Methods. The gene for encoding hTRT catalytic domain was cloned based on RT PCR amplification from HeLa cells and sequenced The cloned hTRTcDNA was in frame inserted into His tag fusion expression vector pEK318 The His tag hTRT fusion proteins were purified by Ni NTA chromatography and stained by western blotting Results. An approximately 620bp fragment was generated and cloned into pBluescript SK+between SalI and BamHI sites DNA sequencing showed the isolated fragment was consistent to those reported SDS PAGE present that a 17kDa protein was expressed stably in E coli JM109 harboring pEKTRT344 containing 6×His tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6×His tag and hTRT 243aa was only detectable as 27 kDa band in western blotting Both of fusion proteins were purified by Ni NTA chromatography and showed single band(>95% purifity) in Coomassie Brilliant staining Western blotting confirmed that two proteins could be recognized by the Ni NTA AP conjugate Conclusions. The hTRT catalytic domain was highly conserved The expressed hTRT protein contained recognizable His tag, telomerase specific and strong antigenic epitops, which may be convenient for further investigation

The Inhibitory Effects of Arresten Protein on Tumor Formation

Objective To examine the inhibitory effects of recombinant purified arresten on tumor formation. Methods Purified arresten protein was incubated with human umbilical vein endothelial cells (HUVECs) and HeLa cells in vitro. The effect on proliferation of HUVECs and HeLa cells was examined using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay, and apoptosis of these cells monitored by flow cytometry. The effect on migration of HUVECs and HeLa cells was examined by Boyden chamber. Twenty colon carcinoma-bearing C67BL/6 mice were used to investigate the antitumor effects of arresten protein. The mice were randomly divided into arresten treatment group (n=10) and control group (n=10). The microvessel densities of the tumors were measured by immunohistochemical staining with anti-CD31 monoclonal antibody. Results Arresten inhibited the proliferation and migration of HUVECs in a dose-dependent manner while promoting apoptosis. However, arresten had no significant effects on the proliferation and apoptosis of HeLa cells. The migration of HeLa cells was modestly inhibited by arresten. The arresten treatment group of mice showed no weight loss or unusual behavior during the course of treatment, and the tumor growth was significantly decreased; in contrast, the control group of mice exhibited rapidly growing tumors and cachexia. A dramatically decreased microvessel density in tumor tissues was found in arresten-treated mice compared with that in the control mice. Conclusion Arresten can inhibit tumor growth through inhibition of tumor angiogenesis.

Effects of antisense oligonucleotides targeting VEGF on radio sensitivity of uterine cervix cancer Hela cells

Objective： To determine the impact of antisense oligonucleotides targeting vascular endothelial growth factor （VEGF） on radiosensitivity of uterine cervix cancer Hela cells. Methods： VEGF antisense oligodeoxynucleotides （ASODN） was transfected into Hela cells by liposome-mediated method. Cells transfected with the oligodeoxynuclecotide and saline were used as control groups. Cells were irradiated by 6 MV X ray at the dose of 0 Gy, 2 Gy, 4 Gy and 6 Gy respectively. The expression of VEGF mRNA was determined by RT-PCR. Apoptosis were evaluated using FCM. Cloning efficiency was determined by colony formation assay. Results： The expression of VEGF mRNAwas inhibited by ASODN （P 〈 0.01） in Hela cells. The inhibited activation which was influenced by radiation resulted in increasing apoptosis （P 〈 0.01） and inhibiting plating efficiency （P 〈 0.01）. Conclusion： The expression of VEGF induced by X irradiation in Hela cells can be blocked by VEGF ASODN. Treatment with VEGF might increase apoptosis in HeLa cells and enhance radiosensitivity.

Effects of HMGB1 Expression Suppressed by siRNA on Cell Cycle and Proliferation of Human Cervical Cancer Cell Line HeLa

OBJECTIVE In this study, RNA interference was used to evaluate the effects of HMGB1 expression on cell cycle and proliferation of the human cervical cancer cell line HeLa.METHODS We had previously constructed and screened effective eukaryotic expression vectors carrying PGCsi3.0-1/ HMGB1 siRNA and PGCsi3.0-3/HMGB1 siRNA, then the vectors were transfected into HeLa cells. The expression of HMGB1 before and after transfection in HeLa cells were detected by RT-PCR and Western blot. The cell viability and proliferating activity was tested by Trypan blue dye test and MTT, and the cell cycle was determined bv flow cvtometry.RESULTS The introduction of PGCsi3.0-1/HMGB1 siRNA and PGCsi3.0-3/HMGB1 siRNA inhibited the expression of HMGB1 mRNA and protein efficiently and specifically, there was a significant difference between the siRNA groups and the control groups （P 〈 0.05）. The proliferation speed of PGCsi3.0-1 group and PGC si3.0-3 group were obviously slower than those of PGCsi3.0- Neg group and non-transfected group. Flow cytometry showed that the content of DNA in G2 phase in PGCsi3.0-1 group and PGCsi3.0-3 group were obviously more than those in PGCsi3.0- Neg group and non-transfected group, but the content in S phase was less （P 〈 0.01）. The progression of cell cycle was arrested from G2 to S phase.CONCLUSION PGCsi3.0-1/HMGB1 siRNA and PGCsi3.0-3 /HMGB1 siRNA could specially suppress the expression of HMGB1 gene, inhibit the proliferation speed of HeLa cells effectively, and arrest the progression of cell cycle from G2 to S phase. RNAi provides a new approach to the bio-therapy of cervical cancer.

SENSITIZATION OF ACNU KILLING EFFECTS ON HeLa S3 CELLS BY MGMT ANTI SENSERNA TRANSFECTION

O6 -methylguanine- DNA- methyltransferase (MGMT ) plays a very important role in the cellular resis- tance to nitrosoureas drugs. Inhibition of MGMT might be a useful approach in tumor chemotherapy. In this study, the depletion of MGMT activity by retroviral-mediated antisense RNA transfection were reported. Three retroviral vectors expressing MGMT antisense RNA were constructed and transfected into HeLa S3 cells. The difference of MGMT mRNA, MGMT activity as well as cellular resistance to ACNU before and after transfection were observed. It was found that antisense RNA targeting 5’region and whole length of MGMT mRNA could partially deplete MGMT activity and enhance killing effects of ACNU. However, 3’ region antisense RNA had no effect on MGMT modulation.

Epigenetic Repression of SATB1 by Polycomb Group Protein EZH2 in Epithelial Cells

Objective To study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1. Methods HeLa epithelial cells were treated with either histone deacetylase inhibitor (HDACi) trichostatin A (TSA) or DNA methylation inhibitor 5-Aza-C before detecting SATB1 expression. Luciferase reporter system was applied to measure effects of EZH2 on SATB1 promoter activity. Over-expression or knockdown of EZH2 and subsequent quantitative reverse transcription-polymerase chain reaction were performed to determine the effect of this Polycomb group protein on SATB1 transcription. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of EZH2 and trimethylated H3K27 (H3K27me3) at SATB1 promoter in HeLa cells. K562 cells and Jurkat cells, both having high-level expression of SATB1, were used in the ChIP experiment as controls. Results Both TSA and 5-Aza-C increased SATB1 expression in HeLa cells. Over-expression of EZH2 reduced promoter activity as well as the mRNA level of SATB1, while knockdown of EZH2 apparently enhanced SATB1 expression in HeLa cells but not in K562 cells and Jurkat cells. ChIP assay results suggested that epigenetic silencing of SATB1 by EZH2 in HeLa cells was mediated by trimethylation modification of H3K27. In contrast, enrichment of EZH2 and H3K27me3 was not detected within proximal promoter region of SATB1 in either K562 or Jurkat cells. Conclusion SATB1 is a bona fide EZH2 target gene in HeLa cells and the repression of SATB1 by EZH2 may be mediated by trimethylation modification on H3K27.

Influence of CDK1 and CDK2 siRNA interference on tumor cell cycle and cell apoptosis

Objective： We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods： The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results： CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotransfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate ceJls among cotransfected cells were observed under the microscope. Conclusion： The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.

Effects of Res on proliferation and apoptosis of human cervical carcinoma cell lines C33A,SiHa and HeLa

Objective:To explore the effects of Res on the proliferation and apoptosis of human cervical cancer cell lines C33A,SiHa and HeLa.Methods:Inhibition rates by different concentrations of Res were calculated using MTT method.Apoptosis rates and cell cycles were measured and examined by flow cytometry(FCM).Morphological configuration of apoptotic cells were observed under the fluorescence microscope.Results:The growth of cancer cells was inhibited by Res of varied concentrations in a time-and dose-dependent manner(P<0.01).The cells showed characteristic apoptosis morphologic changes under fluorescence microscope.Res exerted no effects on cell cycles.Conclusion:Res inhibits the growth of cervical cell lines C33A,SiHa and HeLa by inducing cell apoptosis in a time-and dose-dependent manner.

Proliferation inhibition of human cervical cancer HeLa cells by Casticin in vitro

Objective： The aim of the study was to investigate the effect of Casticin on proliferation inhibition of human cervical cancer HeLa cells in vitro and to unravel the associated mechanisms. Methods： Human cervical HeLa cells were cultured in vitro. The inhibitory effect of Casticin on the viability of human cervical cancer HeLa ceils was evaluated by the MTT assay. The colony formation ability was detected by plate colony formation assay. Distribution of cell cycle was analyzed by flow cytometry. The protein expression levels were analyzed by Western blot. Results： Casticin significantly inhibited the growth of human cervical cancer HeLa cells in a dose- and time-dependent manner, and the IC50 was 2.82 μg/mL. The colony-forming rate was reduced drastically compared with control group （P 〈 0.05）. The cells were markedly arrested at G2/M phase after the treatment of Casticin for 48 h. Western blot showed that the expression of p21 protein was up-regulated and protein level of Cyctin B1 was depressed by Casticin in a concentration dependent manner. Conclusion： Casticin could inhibit the cell growth and lead to cell arrest in human cervical cancer HeLa cells, and the down-regulation of Cyclin B1 protein expression and activation of p21 protein might contribute to Casticin induced cell arrest in human cervical cancer HeLa cells.

Expression of PADI4 in hepatocellular carcinoma

Objective： The aim of the research was to study peptidylarginine deiminase type 4 （PAD4/PADI4） expression and its tumodgenic mechanism in hepatocellular carcinomas. Methods： Expressions of PADI4 and p53 were investigated in tumors and non-tumor tissues by Western blot in patients with hepatocellular carcinomas. We constructed plasmid of PADI4-Flag and transfected it in Hela cells to investigate the mechanism. Results： Western blot analysis showed higher PADI4 expression in hepatocellular carcinomas than in the surrounding healthy tissues. Furthermore, by Western blot, we detected decreased p53 levels in the tumor tissues of patients with hepatocellular carcinomas compared to surrounding healthy tissues. In Hela cells transfected with PcDNA3.0-Flag-PADI4 plasmid, the expression of p53 decreased obviously. Conclusion： Our results suggest that PADI4 elevated in the tissues of hepatocellular carcinomas and induced tumorigenic by down-regulating p53 expression.

Induction of Apoptosis in HeLa Cells by Methanolic Extract of Litsea cubeba Fruit Residue from Essential Oil Extraction

Anticancer activity in vitro ofLitsea cubeba fruit extracts was investigated, focusing on the fruit residue from essential oil extraction. The methanol extract was fractionated by an Amberlite XAD-7 column. Cell viability, cell proliferation and cell death were determined using conversion of WST-8, BrdU incorporation and measurement of released LDH, respectively. Activation of caspase-3/-7 was detected using Z-DEVD-R substrate and morphological characteristics of apoptotic cells were revealed by DAPI staining. It was found that 80-100% methanol fractions （RME-4B, -5A, -5B and -5C） were effective against HeLa cell viability and also promoted cell death. RME-SA and -5B were highly effective in suppressing DNA replication （IC50 4.89 and 3.26 g/mL at 48 h） and also in activation of caspase-3/-7 （9 and 17 times of untreated population at 12 h）. The presence of apoptotic bodies was clearly observed. The results of this study suggested that L. cubeba fruit residue has remarkable apoptosis induction potential for further use in cancer drug research and for waste management in the essential oil industry.

Inhibition of Aspergillus parasiticus and Cancer Cells by Marine Actinomycete Strains

Ten actinomycete strains isolated from the Yellow Sea off China＇s coasts were identified as belonging to two genera by 16S rDNA phylogenetic analysis： Streptomyces and Nocardiopsis. Six Streptomyces strains （MA10, 2SHXF01-3, MA35, MA05-2, MA05-2-1 and MA08-1） and one Nocardiopsis strain （MA03） were predicted to have the potential to produce aromatic polyketides based on the analysis of the KSa （ketoacyl-synthase） gene in the type II PKS （polyketides synthase） gene cluster. Four strains （MA03, MA01, MA10 and MA05-2） exhibited significant inhibitory effects on mycelia growth （inhibition rate 〉50%） and subsequent aria- toxin production （inhibition rate 〉75%） of the mutant aflatoxigenic Aspergillus parasiticus NFRI-95. The ethyl acetate extracts of the broth of these four strains displayed significant inhibitory effects on mycelia growth, and the IC50 values were calculated （MA03： 0.275 mg mL-1, MA01：0.106 mg mL-1, MA10：1.345 mg mL-1 and MA05-2：1.362 mg mL-1）. Five strains （2SHXF01-3, MA03, MA05-2, MA01 and MA08-1） were selected based on their high cytotoxic activities. The ethyl acetate extract of the Nocardiopsis strain MA03 was particularly noted for its high antitumor activity against human carcinomas of the cervix （HeLa）, lung （A549）, kidney （Caki-1） and liver （HepG2） （IC50： 2.890, 1.981, 3.032 and 2.603 μgmL-1, respectively）. The extract also remarkably inhibited colony formation of HeLa cells at an extremely low concentration （0.5μgmL 1）. This study highlights that marine-derived actinomycetes are a huge resource of compounds for the biological control of aflatoxin contamination and the development of novel drugs for human carcinomas.

Protective effects of Rad23 protein on ultraviolet damage to HeLa cells

Protein Rad23, a nucleotide excision repair factor, mainly involves in repairing the DNA damage from environment, such as UV light. The function of Rad23 protein involved in DNA damage repair from many environmental factors has been studied extensively, but it is not clear from ultraviolet irradiation. To further investigate the photo-protective function of Rad23 protein on HeLa cells damaged from UV light irradiation, firstly, HeLa cells were irradiated by UV light and incubated with the fusion protein of pCold-Rad23, then the cell viability and apoptosis rate were detected by MTT and Hoechst33342/Pl fluorescent staining, respectively. The results show that the recombinant Rad23 protein can protect the HeLa cells from UV irradiation, and inhibit the apoptosis of HeLa cell by UV irradiation.

Cell-cycle-dependent variation in UV absorption spectrum of Hela cells treated with Trichostatin A

Objective： The aim of our study was to discovery the different cell cycle arrest effect after different densities HeLa cells treated with Trichostatin A （TSA）. In addition, this study would find some important relationship between cycle arrest effect and UV absorption spectrum of cell. Methods： 0.2 IJM TSA was applied to act on HeLa cells of different density. Then, the cycle arrest effect and UV absorption spectrum of cells were investigated, which provide support to analyze the effect of TSA on cancer cells. Results： Cell cycle arrest effect in G0/G1 of the lower density cells was more obvious than that in other groups. The other discovery in this work was that the cellular UV absorption value was higher when the density of cultured cell was lower. Conclusion： This experiment would guide the clinical study on early or late stage cancer patients in the future. On the other hand, this work indicates when cells were arrested in G0/G1 phase, the cellular absorption value increased at the same time, so UV absorption spectrum could characterize the change of cell cycle.

A new ketosteroid from red alga Acanthophora spicifera

A new ketosteroid, along with six known steroids, was isolated from the ethanolic extracts of red alga Acanthophora spicifera （Vahl.） Boergesen. The structures, identified using chemical and spectroscopic methods including 2D NMR, were： （1） 22-hydroxy-5α-eholest-3,6-dione, （2） 6-hydroxycholest-4-ene-3-one, （3） cholest-4-ene-3,6-dione, （4） cholest-5-ene-3β-ol, （5） 5u-cholestane-3,6-dione, （6） β-Sitosterol and （7） Saringosterol. The MTT method was used to test the cytotoxicity of the compounds against the human cancer cell lines, HCT-8, Bel-7402, BGC-823, A549 and HELA. Compounds 1, 2, 3 and 5 showed moderate cytotoxic activity against human cancer cell lines.