白藜芦醇通过下调PRMT5表达抑制肝胆管癌SMMC-7721细胞的增殖、侵袭和细胞周期

目的:探究白藜芦醇(Res)通过调控PRMT5表达对肝胆管癌SMMC-7721细胞增殖、侵袭、细胞周期的影响及其机制。方法:常规培养正常肝细胞LO2和SMMC-7721细胞,用0、20、40、80μmol/L的Res进行处理,用qPCR法、MTT法、Transwell实验、流式细胞术和WB法分别检测Res处理后PRMT5 mRNA在LO2和SMMC-7721细胞中的表达,Res对SMMC-7721细胞增殖能力、侵袭能力、细胞周期和凋亡,以及PRMT5、cyclin D1和cyclin E1蛋白表达的影响。结果:PRMT5在SMMC-7721细胞中呈高表达(P<0.01);20、40、80μmol/L Res均能明显抑制PRMT5 mRNA和蛋白在SMMC-7721细胞中的表达(均P<0.01),抑制SMMC-7721细胞的增殖能力(P<0.01)和侵袭能力(P<0.05),阻滞SMMC-7721细胞周期于G0/G1期并促进其凋亡(P<0.01),明显抑制SMMC-7721细胞中周期蛋白cyclin D1、cyclin E1蛋白的表达(P<0.01)。结论:PRMT5在SMMC7721细胞中呈高表达,Res可有效抑制SMMC-7721细胞的增殖和侵袭能力并诱导其凋亡,其机制可能与抑制PRMT5表达相关。

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells

AIM: To study the function of α-fetoprotein (AFP) in SMMC-7721 hepatoma cells.METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescenceactivated cell sorter (FACS).RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis.CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.

Positional and expressive alteration of prohibitin during the induced differentiation of human hepatocarcinoma SMMC-7721 cells

AIM： To explore the existence and distribution of prohibitin （PHB） in nuclear matrix and its co-localization with products of some related genes during the differentiation of human hepatocarcinoma SNMC-7721 cells. METHODS： The nuclear matrix of the SMMC-7721 cells cultured with or without 5 × 10^-3 mmol/L hexamethylene bisacetamide （HNBA） was selectively extracted. Western blot was used to analyze the expression of PHB in nuclear matrix; immunofluorescence microscope observation was used to analyze the distribution of PHB in cell. LCSM was used to observe the co-localization of PHB with products of oncogenes and tumor suppressor genes. RESULTS： Western blot analysis showed that PHB existed in the composition of nuclear matrix proteins and was down-regulated by HMBA treatment. Immunofluorescence observation revealed that PHB existed in the nuclear matrix, and its distribution regions and expression levels were altered after HMBA treatment. Laser scanning confocal microscopy revealed the co-localization between PHB and the products of oncogenes or tumor repression genes including c-los, c-myc, p53 and Rb and its alteration of distributive area in the cells treated by HMBA. CONCLUSION： These data confirm that PHB is a nuclear matrix protein, which is located in the nuclear matrix, and the distribution and expression of PHB and its relation with associated genes may play significant roles during the differentiation of SMMC-7721 cells.

Apoptosis of hepatoma cells SMMC-7721 induced by Ginkgo biloba seed polysaccharide

AIM: To study the apoptosis of hepatoma cells SMMC-7721induced by polysaccharide isolated from Ginkgo biloba seed.METHODS: Ginkgo biloba seed polysaccharide (GBSP) wasisolated by ethanol fractionation of Ginkgo biloba seed andpurified by Sephadex G-200 chromatography. The purity ofGBSP was verified by reaction with iodine-potassium iodideand ninhydrin and confirmed by UV spectrophotometer,cellulose acetate membrane electrophoresis and Sepharose4B gel filtration chromatography. The Scanning ElectronMicroscope (SEM) and Flow Cytometrv (FCM) were used toexamine the SMMC-7721 cells with and without GBSPtreatment at 500 mg/ml for 36 h.RESULTS: GBSP product obtained was of high purity withthe average molecular weight of 1.86 × 105. Quantitativeanalysis of SMMC-7721 cells in vitro with FCM showed thatthe percentages of G2-M cells without and with GBSPtreatment were 17.01±1.28 % and 11.77±1.50% (P＜0.05),the debds ratio of the cells were 0.46±0.12 % and 0.06±0 .06 %(P＜0.01), and the apoptosis ratio of cells was 3.84±0 .55 %and 9.13±1.48 %(P＜0.01) respectively. Following GBSPtreatment, microvilli of SMMC-7721 cells appeared thinnerand the number of spherical cells increased markedly. Mostsignificantly, the apoptosis bodies were formed on andaround the spherical cells treated with GBSP.CONCLUSION: GBSP could potentially induce the apoptosisof SMMC-7721 cells.

Effects of cytotoxic T lymphocytes on hepatoma cell line SMMC-7721 induced by different subsets of dendritic cells in vitro

BACKGROUND: Dendritic cells (DCs) loaded with complex antigen are always used to induce cytotoxic T lymphocytes (CTLs) which have a specific anti-tumor activity. However, CTLs can assault autologous cells induced by DCs loaded with autologous antigen. This study aimed to explore how to weaken the autoimmune reaction induced by DC vaccine by combining mature DC (mDC) activating immunity and immature DC (imDC) leading to immune tolerance to make hepatocellular carcinoma (HCC) vaccine in vitro. METHODS: DC progenitors derived from human peripheral blood were assigned to two groups. One was cultured to mDC and pulsed with frozen-thawed antigen (FTA) of human HCC cell line SMMC-7721 cells (mDC group), and the other was cultured to imDC and pulsed with FTA of human liver cell line L-02 cells (imDC group). The morphology of DCs was monitored and cells phenotypes including HLA-DR, CD80, CD1α, CD83 were assayed by flowcytometry (FCM). The concentrations of interleukin-12 (IL-12) in the supernatant were assayed by ELISA. Methyl thiazolyl tetrazolium (MTT) was used to evaluate T cell proliferation induced by mDC and imDC and the killing rate of CTL induced by mDC and imDC respectively/together on SMMC-7721 and L-02 cells. RESULTS: Compared with the imDC group, the mDC group was characterized by the following: increased secretion of IL-12 (P【0.05); higher expression of HLA-DR, CDla, CD80, CD83; and stronger activity in stimulating proliferation of isogenic T cells (P【0.05). CTL induced by the mDC group had a significant killing response to SMMC-7721 as well as a higher killing rate for L-02 (P】0.05). CTL induced by mDC and imDC together had a higher killing response to SMMC-7721, but a lower killing rate for L-02(P【0.01). CONCLUSIONS: CTL induced by mDC and imDC together has a higher antigen-specific killing response in vitro than that induced by mDC alone. This may be of greater clinical value.

Effects of tachyplesin on the regulation of cell cycle in human hepatocarcinoma SMMC-7721 cells

AIM: To investigate the effects of tachyplesin on the cell cycle regulation in human hepatcarcinoma cells.METHODS: Effects of tachyplesin on the cell cycle in human hepatocarcinoma SMMC-7721 cells were assayed with flow cytometry. The protein levels of p53, p16, cyclin D1 and CDK4 were assayed by immunocytochemistry. The mRNA levels of p21WAF1/CIP1 and c-myc genes were examined with in situ hybridization assay.RESULTS: After tachyplesin treatment, the cell cycle arrested at G0/G1 phase, the protein levels of mutant p53, cyclin D1 and CDK4 and the mRNA level of c-myc gene were decreased, whereas the levels of p16 protein and p21wWF1/CIP1 mRNA increased.CONCLUSION: Tachyplesin might arrest the cell at G0/G1 phase by upregulating the levels of p16 protein and p21WAF1/CIP1 mRNA and downregulating the levels of mutant p53, cyclin D1 and CDK4 proteins and c-myc mRNA, and induce the differentiation of human hepatocacinoma cells.

Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells

AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7,-9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation ofhuman hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.

Effects of tachyplesin on proliferation and differentiation of human hepatocellular carcinoma SMMC-7721 cells

AIM: To investigate the antitumor activities of tachyplesin on human hepatocellula r carcinoma (HCC) cells.METHODS: Tachyplesin, isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes, was used to treat the human HCC cell line SMMC-7721. Effects of tachyplesin on the proliferation of SMMC-7721 cells were measured with trypan blue dye exclusion test and HE staining. The morphology and ultrastructure of the cells were examined by light microscopy and transmission electron microscopy, respectively. The activities of γ-glutamyltransferase (γ-GT) and tyrosine aminotransferase (TAT) were assayed with biochemical methods. The levels of alpha fetoprotein (α-FP), proliferating cell nuclear antigen (PCNA), p21wAF1/CIP1 and c-myc were examined by immunocytochemistry. RESULTS: After treatment with tachyplesin 3.0 mg/L, the proliferation of SMMC-7721 cells was inhibited significantly, with the cell growth inhibitory rate amounted to 55.57 % and the maximum cell mitotic index declined by 43.68 %. The morphology and ultrastructure underwent restorational alteration. The activity of γ-GT declined while TAT activity increased obviously, and the levels of α-FP and PCNA decreased. Moreover, the expression of p21WAF1/CIP1 protein was upregulated and that of c-myc protein was down-regulated. CONCLUSION: Tachyplesin could effectively inhibit the proliferation of hepatocarcinoma cells, reverse the malignant morphological and ultrastructural characteristics, alter the levels of enzymes and antigens, regulate the expression of differentiation-associated oncogene and tumor suppressor gene, and induce hepatocarcinama cell differentiation.

Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis

AIM: To investigate the in vitro anti-hepatocellular carcinoma (HCC) activity of docetaxel against SMMC-7721 HCC cells and its possible mechanism.METHODS: The HCC cells were given different concentrations of docetaxel and their growth was measured by colony forming assay. Cell cycle and apoptosis were analyzed by flow cytometry and fluorescence microscopy (acridine orange/ethidium bromide double staining, AO/EB), as well as electronic microscopy. The SMMC-7721 HCC cell reactive oxygen species (ROS) and glutathione (GSH) were measured after given docetaxel.RESULTS: Docetaxel inhibited the hepatocellular carcinoma cells growth in a concentration dependent manner with IC505×10-10 M. Marked cell apoptosis and G2/M phase arrest were observed after treatment with docetaxel ≥10-8M.Docetaxel promoted SMMC-7721 HCC cells ROS generation and GSH deletion.CONCLUSION: Docetaxel suppressed the growth of SMMC7721 HCC cells in vitro by causing apoptosis and G2/M phase arrest of the human hepatoma cells, and ROS and GSH may play a key role in the inhibition of growth and induction of apoptosis.

Serum deprivation enhances DNA synthesis of human hepatoma SMMC-7721 cells

AIM To determine the relationship between serum deprivation or serum levels and cell proliferation of human hepatoma SMMC 7721 cells. METHODS Human hepatoma SMMC 7721 cells were grown in RPMI 1640 supplemented with 10% fetal calf (FCS) in 5% CO 2 incubator at 37℃ for 24h , and culture media were replaced to serum free or different serum FCS levels (2 5%, 5%, 10%, 20% and 25%). Six h, 12h , 18h and 24h after the culture, the cells were incorporated TdR for 4h . At last TdR incorporation was detected with liquid scintillation counting. RESULTS DNA synthesis of SMMC 7721 cells could be sharply stimulated by short time (6h) serum deprivation (the cpm value of 3H TdR incorporation of cells in serum free was 39 32 fold higher than cells in 25% serum), and the incorporation of 3H TdR was negatively related to the serum levels. Longer time serum starvation ( 12h , 18h and 24h ) also greatly stimulated DNA synthesis, although the cpm value of 3H TdR incroporation was less than that in 6h serum deprivation. Morphology of cells cultured in different serum levels also showed significant difference. CONCLUSIONS Compared with other cell lines such as BEL7404 and Swiss 3T3, human hepatoma SMMC 7721 cells had different response to the serum deprivation. Short time serum deprivation could greatly stimulate DNA synthesis of human hepatoma SMMC 7721 cells. Precautions must be given to the changes of serum levels for the detection of growth factors and drugs using SMMC 7721 cells as a model.

Activity Determination of 8 Chinese Herbs against Hepatoma Cell SMMC-7721 in Vitro by MTT Method

[Objective] The aim was to build up a set of efficient and rapid models for laboratory to screen anti-hepatocellular carcinoma active substance in vitro. [ Method] By using MTT method, the activities of anti-hepatocellular carcinoma SMMC-7721 in vitro from Cymbopogon distans, Lobelia chinensis, Buddleja offlcinalis, Glycyrrhiza uralensis, Sanguisorba officinalis, Bupleurum chinense, Apium graveolen and Curuma zedoaria were tested. The growth curve of hepatoma cell was described, and the growth status in different periods were observed by inverted microscope. [ Result] Cells induced by active substance would be condensing, clear brim, which have significant differences from normal SMMC- 7721 cells. The results suggested that ESCG, ESCC, ESCB could inhibit proliferation of SMMC-7721 cells at the concentration of 1.0 -1.5 mg/ml, and the inhibition rate were 51.6%, 48.5%, 52.9% respectively. With the increasing of concentration, the inhibition strengthened. [ Conclusion] MTT method could be used as a basic model for screening important anti-hepatoma.

当归、红芪提取物对人肝癌SMMC-7721细胞辐射增敏作用

目的:探讨当归、红芪提取物对直线加速器(X射线)辐射后人肝癌细胞SMMC-7721的增敏作用。方法:96孔板培养人肝癌细胞SMMC-7721,设9个组,对照组(N)即正常对照组,纯辐射组(F)给以源皮距100cm 4Gy的X射线,另设3个纯药物组(DH),设高(H:8mg/mL)、中(M:6mg/mL)、低(L:4mg/mL)三个药物浓度梯度,3个辐射加药组(F+DH)和1个空白组(只有培养液无细胞)。每组设6个复孔。采用MTT法计算当归、红芪提取物对人肝癌SMMC-7721细胞辐射前后增殖活性的影响。流式细胞术检测不同组细胞的周期情况,透射电镜及倒置显微镜分别观察各组细胞结构变化,用蛋白印记法检测不同组细胞中p21蛋白表达情况。结果:MTT法证明当归、红芪提取物具有抑制人肝癌细胞SMMC-7721增殖作用,对于辐射后的肝癌细胞具有很好的周期抑制作用。不同剂量当归、红芪提取物作用于细胞24h后,抑制人肝癌细胞SMMC-7721增殖呈剂量依赖关系,药物浓度越高,抑制作用越显著。SMMC-7721细胞经辐射后,细胞的抑制率为37.58%,辐射后加不同浓度当归、红芪提取物(4mg/mL、6mg/mL、8mg/mL),联合作用对细胞的抑制率升高,分别为45.27%、51.86%、59.34%,且呈药物浓度依赖关系,差异具有统计学意义(P<0.01)。辐射组肝癌细胞较正常组细胞G0/G1期细胞数明显增多,由44.70%升至71.60%(P<0.01),S期细胞减少,高浓度药物也可以使G0/G1期细胞比例有所升高(P<0.01)。辐射加药组细胞G0/G1期阻滞最为明显,并呈药物浓度梯度关系。高浓度药物组、辐射组细胞p21蛋白表达较正常组升高,辐射组+高浓度药物组细胞p21蛋白表达较辐射组明显升高。结论:当归、红芪提取物对于人肝癌SMMC-7721细胞具有很好的抑制作用,对经X线辐射后的细胞具有很好的增敏作用。

圣草次苷抑制肝细胞癌SMMC-7721细胞的增殖和迁移:基于激活ROS/MAPKs信号轴

目的探讨圣草次苷通过ROS/MAPKs信号轴调控肝癌SMMC-7721细胞增殖和迁移的作用机制。方法分别用0、25、50、100、150、200、250、300μg/mL圣草次苷处理肝癌SMMC-7721细胞系24 h后,CCK-8法检测细胞活性;不同浓度的圣草次苷(100、200、300μg/mL)处理细胞后,运用划痕实验分别在24、48、72 h检测划痕愈合率,Transwell实验检测细胞迁移率,克隆形成实验检测细胞增殖能力,DAPI染色观察细胞核形态变化,Western blot检测侵袭蛋白E-cadherin、N-cadherin、MMP-2、MMP-9、凋亡因子PARP和Pro-caspase 3以及MAPKs通路p-JNK、p-P38、p-ERK信号分子;用(分别为2、5、10μmol/L)ERK抑制剂U0126、JNK抑制剂SB203580和P38抑制剂SP600125预先处理细胞2 h,再用200μg/mL圣草次苷处理细胞24 h,Western blot检测凋亡蛋白PARP的切割情况;分别用N-乙酰-半胱氨酸(NAC)(30μmol/L)或圣草次苷(100、200、300μg/mL)单独或共处理细胞后,通过DCFH-DA荧光探针法分别在15、30、60 min检测内源性活性氧(ROS)水平。结果圣草次苷在50μg/mL范围内,对细胞活力基本无影响(P>0.05);圣草次苷能够显著抑制SMMC-7721细胞的划痕愈合(P<0.01),细胞迁移(P<0.01)和克隆形成(P<0.01),在300μg/mL浓度时作用最显著;圣草次苷明显减少侵袭蛋白N-cadherin、MMP-2、MMP-9的蛋白表达量(P<0.01),上调E-cadherin的蛋白表达量(P<0.05);圣草次苷诱导SMMC-7721细胞发生显著的凋亡形态学变化,Pro-caspase 3表达降低,PARP切割增多(P<0.01);圣草次苷在300μg/mL刺激30 min时ROS表达水平较高,NAC(30μmol/L)处理后,细胞内ROS表达水平下降明显;圣草次苷能够显著诱导MAPKs信号途径JNK、P38和ERK的磷酸化(P<0.01),U0126和SB203580能够增强圣草次苷诱导的PAPR切割,而SP600125逆转圣草次苷诱导的PARP切割。结论圣草次苷通过促进细胞内ROS的合成,诱导MAPKs信号通路的活化,调控肝癌细胞系SMMC-7721的增殖、迁移和凋亡。

miR-3682-3p通过调控PI3K/AKT信号通路影响SMMC-7721人肝癌细胞的增殖、侵袭和凋亡

目的分析miR-3682-3p与PI3K/AKT信号通路在肝细胞癌发展中的关系,以期为肝细胞癌的治疗提供新的思路。方法通过qRT-PCR分析MIHA人正常肝细胞和SMMC-7721人肝癌细胞中miR-3682-3p与PI3K/AKT的表达水平。通过转染不同质粒将SMMC-7721人肝癌细胞分为miR-3682-3p mimic组、miR-3682-3p mimic NC组、miR-3682-3p inhibitor组和miR-3682-3p inhibitor NC组,采用双荧光素酶报告基因实验分析miR-3682-3p与PI3K的相互作用关系。采用蛋白免疫印迹、qRT-PCR、流式细胞术、细胞划痕和Transwell实验分析不同实验组细胞的增殖、侵袭和凋亡情况。结果miR-3682-3p能够靶向调控PI3K/AKT信号通路,miR-3682-3p表达能够激活PI3K/AKT信号通路,肝癌细胞的增殖、迁移和侵袭能力增强,凋亡减少。而抑制miR-3682-3p表达后,PI3K/AKT通路受到抑制,肝癌细胞的增殖、迁移和侵袭能力减弱,凋亡增加。结论PI3K是miR-3682-3p的直接靶点,miR-3682-3p通过激活PI3K/AKT通路促进SMMC-7721人肝癌细胞的体外转移活性。

Protein profile of human hepatocarcinoma cell line SMMC-7721:Identification and functional analysis

AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS： Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis （2DE）. The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION： Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

WWOX induces apoptosis and inhibits proliferation of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721. METHODS: Full-length WWOX cDNA was amplified from normal human liver tissues. Full-length cDNA was subcloned into pEGFP-N1, a eukaryotic expression vector. After introduction of the WWOX gene into cancer cells using liposomes, the WWOX protein level in the cells was detected through Western blotting. Cell growth rates were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Cell cycle progression and cell apoptosis were measured by flow cytometry. The phosphorylated protein kinase B (AKT) and activated fragments of caspase-9 and caspase-3 were examined by Western blotting analysis. RESULTS: WWOX significantly inhibited cell proliferation, as evaluated by the MTT and colony formation assays. Cells transfected with WWOX showed significantly higher apoptosis ratios when compared with cells transfected with a mock plasmid, and overexpression of WWOX delayed cell cycle progression from G1 to S phase, as measured by flow cytometry. An increase in apoptosis was also indicated by a remarkable activation of caspase-9 and caspase-3 and a dephosphorylation of AKT (Thr308 and Ser473) measured with Western blotting analysis. CONCLUSION: Overexpression of WWOX induces apoptosis and inhibits proliferation of the human hepatic carcinoma cell line SMMC-7721.

Subcellular daunorubicin distribution and its relation to multidrug resistance phenotype in drug-resistant cell line SMMC-7721/R

AIM: To investigate the correlation between subcellulardaunorubicin distribution and the multidrug resistancephenotype in drug-resistant cell line SMMC-7721/R.METHODS: The multidrug resistant cell line SMMC-7721/R, a human hepatocellular carcinoma cell line,was established. Antisense oligonudeotides (AS-ODN)were used to obtain different multidrug resistancephenotypes by inhibiting the expression of mdr1 geneand/or multidrug resistance-related protein gene(mrp)using Lipofectamine as delivery agent. Expression ofmdr1 and mrp genes was evaluated by RT-PCR andWestern blotting. Intracellular daunorubicin (DNR)concentration was measured by flow cytometry.Subcellular DNR distribution was analyzed by confocallaser scanning microscopy. Adriamycin (ADM) and DMRsensitivity was examined by MTT method.RESULTS: Low level expression of mdr1 and mrp mRNAsand no expression of P-Glycoprotein(P-gp) andmultidrug resistance-related protein (P190) weredetected in parental sensitive cells SMMC-7721/S, butover-expression of these two genes was observed indrug-resistant cell SMMC-7721/R. The expression ofmdr1 and mrp genes in SMMC-7721/R cells was downregulated to the level in the SMMC-7721/S cells by AS-ODN. Intracellular DNR concentration in SMMC-7721/S cells was 10 times higher than that in SMMC-7721/Rcells. In SMMC7721/S cells intracellular DNRdistributed evenly in the nucleus and cytoplasm, whilein SMMC-7721/R cells DNR distributed in a punctatepattern in the cytoplasm and was reduced in thenucleus. DNR concentration in SMMC-7721/R cells co-transfected with AS-ODNs targeting to mdr1 and mrpmRNAs recovered to 25 percent of that in SMMC7721/Scells. Intracellular DNR distribution pattern in drug-resistant cells treated by AS-ODN was similar to drug-sensitive cell, and the cells resistance index (RI) to DNRand ADM decreased at most from 88.0 and 116.0 to 4.0and 2.3, respectively. Co-Transfection of two AS-ODNsshowed a stronger synergistic effect than separatetransfection.CONCLUSIONS: P-gp and P190 are two membersmediating MDR in cell line SMMC7721/R. Intracellulardrug concentration increase and subcellular distributionchange are two important factors in multidrugresistance (MDR) formation. The second factor, drugstransport by P-gp and P190 from cell nucleus to organellin cytoplasm, may play a more important role.

Over-expression of LPTS-Lin hepatocellular carcinoma cell line SMMC-7721 induces crisis

AIM: To evaluate the function of the longer transcripts LPTS-Lin hepatocellular carcinoma cell line SMMC-7721.METHODS: SMMC-7721 cells were transfected with LPTSL expression construct and stably transfected cells were selected by G418. Multiple single clones formed and were checked for their phenotype. In the study of the effect on telomerase activity of LPTS-Lin vitro, GST-LPTS-L fusion protein was expressed in E.coli and purified by glutathioneagarose column. Telomeric repeat amplification protocol (TRAP) assays were performed to study the influence of telomerase activity in SMMC-7721 cells.RESULTS: Over-expression of LPTS-L induced SMMC-7721 cells into crisis. LPTS-L could inhibit the telomerase activity in SMMC-7721 cellsin vitro.CONCLUSION: LPTS-L is a potent telomeraseinhibitor. Over-expression of LPTS-L can induce hepatoma cells into crisis due to the reduction of telomerase activity.

Expression of angiostatin cDNA in human hepatocellular carcinoma cell line SMMC-7721 and its effect on implanted carcinoma in nude mice

AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice.METHODS: A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed. Then pcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine. The resistant clone was screened by G418 filtration and identified by RT-PCR and Western blotting. Nude mice were divided into three groups of 10 each. Mice in blank control group were only injected with SMMC-7721 cells. Mice in vector control group were injected with SMMC-7721 cells transfected with pcDNA3.1 (+) vector,whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid.Volume, mass and microvessel density (MVD) of the tumors in different groups were measured and compared.RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line.No significant difference was observed in the growth speed of SMMC-7721 cells between groups transfected with and without angiostatin cDNA. Tumor volume, mass and MVD in the angiostatin group were significantly lower than those in the blank control group and vector control group (P<0.01).The inhibitory rate of tumor reached 78.6%. Mass and MVD of the tumors only accounted for 34.6% and 48.9% respectively of those in the blank control group.CONCLUSION: Angiostatin cDNA could be stably expressed in human hepatocellular carcinoma cell line SMMC-7721 without obvious inhibitory effects on the growth of SMMC-7721 cells. When implanted into nude mice, SMMC-7721 cells transfected with angiostatin cDNA show a decreased tumorigenic capability. It suggests that angiostatin can inhibit tumor growth through its inhibition on angiogenesis in tumors.