Inhibition of VEGF expression and SMMC 7721 cell growth by VEGFsiRNA

AIM: To determine the inhibition of VEGF small interfering RNA (siRNA) on expression of VEGF protein and SMMC 7721 cell growth. METHODS: Nine VEGFsiRNA sequences with nineteen nucleotides were designed under the assistance of computer. The inhibitory effect of VEGFsiRNA was analyzed by CCK8. The protein level of VEGF in the media was determined by ELISA. The change of cell cycle was detected by flow cytometry. RESULTS: All the VEGFsiRNA were capable of inhibiting the proliferation of SMMC 7721 cells significantly compared with control and lipofectamine control, while the inhibitory effects of VEGFsiRNA2, VEGFsiRNA4, VEGFsiRNA6, VEGFsiRNA7, VEGFsiRNA8 and VEGFsiRNA9 were better than that of antisense oligodeoxynucleotide. All the VEGFsiRNA reduced the expression of VEGF protein. The effect of VEGFsiRNA7 and VEGFsiRNA2 were the best with the inhibitory rates of 52.65% and 50.43%， respectively. VEGFsiRNAs induced the S arrest in SMMC 7721 cells. The signs of apoptosis in SMMC 7721 cells induced by VEGFsiRNA were not observed. CONCLUSION: VEGFsiRNA sequences were designed and synthesized successfully. VEGFsiRNA effectively inhibited the proliferation of SMMC 7721 cells and reduced the level of VEGF protein.     

Inhibition of human hepatocellular carcinoma cell proliferation by galactose receptor mediated c-myc antisense oligonucleotide

AIM: To evaluate the inhibitory effect of galactose (Gal)-polyethyleneimine (PEI)-c-myc antisense oligodeoxynucleotide (ASODN) complex on proliferation of human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cell line Bel-7402 was treated with Gal-PEI-ASODN complex. Cell proliferation was tested by trypan blue dye at different time points and with various concentrations of ASODN treatment. Cell morphology was observed under inverted microscope, cell hypodiploid percentage was analyzed by flow cytometry and cell ultrastructure was observed through electron microscopy. RESULTS: Compared with ASODN group (20 μmol/L) from 0 h to 96 h, Gal-PEI-ASODN complex (with ASODN 0.75 μmol/L) significantly suppressed Bel-7402 cells proliferation, the ASODN concentration within Gal-PEI-ASODN complex and time course acquired were significantly lower and shorter, respectively. Incubated with pure ASODN at different concentrations for 72 hours, cell proliferation was inhibited and IC₅₀ was 20.9 μmol/L; while mediated with galactose receptor for 48 hours, ASODN significantly inhibited cell proliferation and IC₅₀ was only 0.294 μmol/L, the inhibitory efficacy of ASODN enhanced 70.9 folds. While Bel-7402 cells were incubated with Gal-PEI-ASODN complex for 48 hours, cell hypodiploid percentage was much higher than ASODN groups and cell apoptosis was seen under electron microscopy. CONCLUSIONS: Galactose receptor mediated ASODN delivery may significantly increase proliferation inhibition efficacy on Bel-7402 cells.     

Treatment of hepatocellular carcinoma in vitro and in vivo with yCD/TK double suicide gene driven by AFP promoter

AIM: To study the effect and mechanism of yeast cytosine deaminase/ thymidine kinase (yCD/TK) double suicide gene driven by alpha fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) in vitro and in vivo. METHODS: The expression plasmid with yCD/TK double suicide gene, which was driven by AFP promoter, was constructed. HepG2 (AFP positive) and SMMC7721 (AFP negative) human HCC cell lines were both transfected with the above-mentioned expression plasmid through cationic liposome. The cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The cell proliferation and cell cycle phase were evaluated by MTT test and flow cytometry respectively. The effect of double suicide gene on HCC xenografts in nude mice was observed through measuring the tumor size and the number of apoptosis cells. RESULTS: The double suicide gene was expressed selectively on HepG2 cells, rather than on SMMC7721 cells. The 5-FC and/or GCV inhibited effectively the proliferation of HepG2 cells in a dose-dependent manner, but had no influence on SMMC7721 cells. The inhibitory effect on HepG2 cells among different treatments was GCV+5-FC>5-FC>GCV. In vivo, the treatments inhibited markedly the growth of HepG2 cell xenografts in nude mice, transfected with yCD/TK gene. More apoptotic cells were found in HepG2 xenografts after the treatment. However, the growth of SMMC7721 cell xenografts could not be inhibited by this double suicide gene therapy, and few apoptotic cells were found. CONCLUSION: yCD/TK double suicide gene driven by AFP promoter has a significant efficacy in treatment of AFP positive HCC. Cell apoptosis may be an important mechanism of yCD/TK double suicide gene-inhibiting the growth of HCC.     

Inhibitory effect of liposomes survivin antisense oligonucleotide on human hepatic carcinoma transplanted subcutaneously in nude mice

AIM: To explore the effects of liposomes survivin antisense oligonucleotides (ASODN) on growth of human hepatic carcinoma transplanted subcutaneously in nude mice. METHODS: Nude mouse model of human hepatic cancer was established by transplantation of hepatic cancer cell line SMMC-7721/ADM subcutaneously. Models were divided randomly into six groups: control group, liposome group, sense oligonucleotide (SODN) group, 200 μg/L, 400 μg/L and 600 μg/L ASODN groups. Different treatments were given respectively. Weight and volume of subcutaneous tumors were measured, and tumor growth inhibitory rate was calculated. Morphological changes of transplanted tumor cells were observed under light microscope. The expression of Survivin was detected by immunohistochemistry. RESULTS: The growth of tumors was significantly inhibits in all ASODN groups compared with control, liposome and SODN groups (P<0.05). Volume of subcutaneous tumors decreased in a time-dependent and dosage-dependent manner (P<0.5). CONCLUSION: Survivin ASODN inhibits the growth of human hepatic carcinoma in nude mice.     

Role of VEGF-C in lymphangiogenesis and lymphatic metastasis of breast cancer

AIM: To study the inhibitory effect of VEGF-C/Flt-4 system on lymphangiogenesis and lymphatic metastasis of breast cancer. METHODS: Lymphatic endothelial cells (LEC) were cultured in vitro, the effects of VEGF-C and anti-Flt-4 antibody on the proliferation of treated cells were observed. The antisense oligodeoxynucleotides (ASODN) targeting VEGF-C was designed and its effect on VEGF-C gene expression in vitro experiments was observed. The nude mice transplantation tumor model was made and the effects of VEGF-C ASODN on lymphangiogenesis and metastasis in the model were determined. RESULTS: The supernatant of cultured PC3 cells promoted LEC proliferation obviously while the cells treated with anti-Flt-4 antibody were obviously decreased whenever cell counting. The mRNA and protein expression of VEGF-C in MCF-7 cells treated with ASODN were significantly lower than that in control groups in vitro. In vivo ASODN also significantly reduced the VEGF-C mRNA expression detected by RT-PCR. The result of 5-Nase-ALPase enzyme -histochemistry showed that ASODN had obvious inhibitory effect on tumor lymphangiogenesis. Tumor growth velocity in ASODN group was much slower than that in control group. ASODN also inhibited tumor volume and lymphatic metastasis. CONCLUSION: The strong relationships between VEGF-C/Flt-4 system and lymphangiogenesis and lymphatic metastasis of breast cancer have been observed. If the expression of Flt-4 is blocked, the proliferation of LEC induced by tumor cells can be blocked in some degree. ASODN inhibits tumor lymphangiogenesis and lymphatic metastasis by down-regulating VEGF-C expressions.     

Effects of new drug of sulfonylurea on the glucose uptake in SMMC7721 cells

AIM: To explore the effects of the new drug of sulfonylurea (1-{4-［2-(3-ethyl- 4-methyl-2-oxo-3-pyrroline-1- carboxamido)ethyl］-phenylsulfonyl}-3-(1, 4-tetramethylene)-urea, BGW) on the glucose uptake and the activation of Akt/PKB in SMMC7721 cells. METHODS: Cultured SMMC7721 cells were divided into control group, glibenclamide group, insulin group, BGW group and BGW+insulin group. Scintillation was used to detect the glucose uptake in SMM7721 cells. The activation of Akt/PKB was tested by Western blotting. RESULTS: Compared to control cells, gibenclamide, insulin, BGW and BGW+insulin significantly increased the glucose uptake (P<0.01), and increased by 22%, 108%, 60% and 400% in SMMC7721 cells, respectively. The new drug of sulfonylurea increased the insulin-stimulated glucose uptake (P<0.01) in SMMC7721 cells. The BGW-stimulated glucose uptake was higher than that with the glibenclamide-stimulated, and increased the insulin-stimulated glucose uptake (P<0.01). The sulfonylurea significantly enhanced the activity of Akt in SMMC7721 cells. CONCLUSION: The new drug of sulfonylurea (BGW) stimulates glucose uptake, increases the insulin-stimulated glucose uptake in SMMC7721 cells, and is a potential anti-hyperglycemic agent.     

Effect of epidermal growth factor receptor antibody on human colon carcinoma

AIM: To observe the effect of monoclonal antibody of epidermal growth factor receptor(EGFR) on human colon carcinoma cell lines. METHODS: Cell counting, growth curve measurement and MTT method were applied in this study to examine the proliferation of cultured cells in vitro when different dosage of EGFR McAb is used to treat LST174 colon carcinoma cell lines. RESULT: The proliferation of cultured human colon carcinoma cells could be significantly inhibited in a dose dependent manner by EGFR antibody Compared with the control group, the cell number was decreased by 61.3% and 33.8% respectively when treated with 0.625 mL/L or 2.5 mL/L of EGFR McAb CONCLUSION: EGFR McAb can inhibit cell growth of human colon carcinoma LST174.     

Effects of marrow stromal cell-mediated microenvironment on human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the effects of marrow stromal HS-5 cells on hepatocellular carcinoma SMMC-7721 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the proliferation, migration and invasion abilities of SMMC-7721 cells were detected by MTT, wound-healing and Transwell assays. After co-culture of SMMC-7721 cells with HS-5 cells in the Transwell chamber, the expression of chemokine CCL5 and its receptor CCR5 at mRNA and protein levels in SMMC-7721 cells was examined by quantitative real-time PCR (qRT-PCR), ELISA or Western blotting. Akt and p-Akt473 protein levels in SMMC-7721 cells treated with PI3K inhibitor LY294002 were observed by Western blotting. RESULTS: HS-5-CM promoted the proliferation, migration and invasion abilities of SMMC-7721 cells. The expression of CCL5 and CCR5 at mRNA and protein levels in SMMC-7721 cells was increased after co-cultured with HS-5 cells. PI3K inhibitor LY294002 inhibited the activation of PI3K-Akt signaling pathway and the secretion of CCL5 in SMMC-7721 cells after co-cultured with HS-5 cells. CONCLUSION: HS-5 cells significantly promote the proliferation, migration and invasion abilities of SMMC-7721 cells. Co-culture of SMMC-7721 cells with HS-5 cells activates PI3K-Akt signaling pathway to increase the secretion of CCL5 in SMMC-7721 cells.     

Effects of 2-deoxy-D-glucose and oxaliplatin on proliferation and apoptosis of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of 2-deoxy-D-glucose (2-DG) or oxaliplatin (L-OHP) alone and the combination of both on the proliferation and apoptosis of hepatoma cell line SMMC-7721 in vitro. METHODS: Human hepatoma cell line SMMC-7721 was treated with 2-DG or L-OHP alone, or both. The inhibitory effect on the proliferation of SMMC-7721 cells was estimated by MTT method. The q value, which represents synergistic effect, was determined. Apoptotic rate and cell cycle were assayed by flow cytometry. The activity of caspase-3 was detected by a caspase-3 activity assay kit. RESULTS: 2-DG or L-OHP at different concentrations inhibited the growth of SMMC-7721 cells obviously and the inhibitory effect on SMMC-7721 cell growth strongly depended on the exposure time and dose. When the 2 drugs worked together, the inhibitory effect was improved (P<0.05). 2-DG induced cell apoptosis and arrested the cells at G₂/M phase. When combined with L-OHP,the 2 drugs induced more severe apoptosis and arrested the cells at S and G₂/M phase. Meanwhile, the activity of caspase-3 increased when the 2 drugs used together. CONCLUSION: 2-DG inhibits the growth of hepatoma cell line SMMC-7721. The combination of 2-DG and L-OHP improves the ability of L-OHP to attack the tumor cells. The mechanism might be related to increasing the activity of caspase-3.     

“中国园艺学会热带南亚热带果树分会成立大会暨首届学术研讨会”会讯

“中国园艺学会热带南亚热带果树分会”是由广东省农业科学院果树研究所、福建省农业科学院果树研究所、仲恺农业技术学院园艺系、中国热带农业科学院南亚热带作物研究所等单位联合申请,并经中国园艺学会第九届六次常务理事会扩大会议讨论同意成立的。经筹委会讨论决定,将于200     

Effect of abnormal expression of miR-141 on malignant biological behaviors of human hepatocarcinoma cells

AIM: To investigate the expression of microRNA-141 (miR-141) in human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal hepatocyte line HL-7702, and to analyze the effect of abnormal expression of miR-141 on the malignant biological behaviors of human hepatocarcinoma cells. METHODS: The RNA from SMMC-7721 cells and HL-7702 cells was extracted. SYBR Green real-time PCR was performed to detect the expression of miR-141. Synthetic miR-141 mimic and its negative control were transfected into the SMMC-7721 cells, and miR-141 inhibitor and its negative control were transfected into the HL-7702 cells by the method of Lipofectamine. After transfection, MTS assay and BrdU-ELISA were employed to evaluate the effect of miR-141 on the cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. The changes of migration ability were investigated by Transwell invasion assay. RESULTS: The expression of miR-141 in the SMMC-7721 cells was significantly lower than that in the HL-7702 cells (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of the SMMC-7721 cells transfected with 25 nmol/L miR-141 mimic was significantly inhibited in a time-dependent manner (P < 0.05). The percentages of G₁ phase cells and early apoptotic rate were significantly increased when miR-141 was up-regulated, but the migration ability was inhibited (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of HL-7702 cells transfected with 50 nmol/L miR-141 inhibitor was significantly increased in a time-dependent manner (P < 0.05). When miR-141 was down-regulated, the percentages of G₁ phase cells and early apoptotic rate were significantly decreased, but the migration ability was enhanced (P < 0.05). CONCLUSION: miR-141 is down-regulated in human hepatocarcinoma cell line. Up-regulation of miR-141 will not only inhibit cell proliferation and migration ability, but also affect the cell cycle and apoptosis of SMMC-7721 cells. miR-141 may function as a tumor suppressor gene during HCC development.     

Induction of apoptosis by c-myc antisense oligonucleotide in osteosarcoma cell MG-63

AIM: To study the induction of apoptosis by c-myc antisense oligonucleotide in osteosarcoma cell (MG-63).METHODS: The designed c-myc antisense oligonucleotide fragment was transfected into human osteosarcoma MG-63 cells. The cell growth and apoptosis were measured by the methods of MTT, FCM, HE staining and transmission electron microscopy.RESULTS: The results showed that the proliferation of human osteosarcoma MG-63 cells was inhibited and apoptotic rate was 37.92% when treated with c-myc antisense oligonucleotide at the does of 10.0 μmol/L for 48 h. c-myc antisense oligonucleotide (10.0 μmol/L) also inhibited the expression of c-myc protein.CONCLUSION: c-myc antisense oligonucleotide is able to induce apoptosis in human osteosarcoma MG-63 cells.     

Inhibitory effects of a cyclooxygenase-2 inhibitor,NS398,on cancer cells

AIM:To study the mechanism of growth inhibitory effect of a selective cyclooxygenase-2 (COX-2) inhibitor,NS-398,on cancer cells.METHODS:The esophageal cancer cell line (EC9706),which expresses COX-2 constitutively,and hepatocellular carcinoma cell line (SMMC7721),which expresses no COX-2,were studied.The cell lines were incubated with NS-398 at doses of 10,20,50,100 μmol/L for 24 h,48 h and 72 h.Antiproliferation effect was measured by ［3H］-TdR incorporation.The cell apoptosis were determined by flow cytometry (FCM) and DNA fragmentation analysis.Survivin was detected by immunocytochemical technique.RESULTS:The growth inhibition was induced by NS398 in a dose- and time-dependent manners in both cell lines.FCM analysis revealed a high sub-G1 cell peak in EC9706 group and agarose electrophoresis showed marked apoptosis ladder pattern.However,no apoptosis was observed in SMMC7721 cells treated with NS-398.The difference of apoptosis percentage in EC9706 and SMMC7721 was (45.23±1.08)% and (3.05±0.15)% (P<0.01).After 24 h incubation with NS-398 at concentration of 100 μmol/L,the expression of survivin was markedly reduced in EC9706,no change was observed in SMMC7721.CONCLUSION:NS-398 suppresses cell growth in cancer cell lines by different mechanism.NS-398 suppresses cell growth and increases apoptosis in the cancer cells that expresses COX-2.     

Antitumor activity of Paecilomyces tenuipes polysaccharide and its mechanism in vitro

AIM:To investigate the antitumor activity and mechanism of Paecilomyces tenuipes polysaccharide(PTPS).METHODS:PTPS-I was obtained by water extraction and alcohol precipitation, and purified by DEAE-cellulose and Sephadex G-100 chromatography. Human erythroleukemia cell line K562, laryngocarcinoma cell line Hep2 and hepatic carcinoma cell line SMMC-7721were co-cultured with PTPS-I or the conditioned medium which prepared with PTPS-I-stimulated human mononuclear cells (PTPS-I-MNC-CM), and the proliferation of tumor cells was determined. The cell counting kit-8 (CCK-8) was used to determine the proliferation of MNCs. The FQ-RT-PCR was applied to investigate the expression of TNF-α and IL-6 mRNA in MNCs. RESULTS:PTPS-I-MNC-CM inhibited the proliferation of K562, Hep2 and SMMC-7721 cells in vitro (P<0.01). Cytotoxicity of PTPS-I against K562, Hep2 and SMMC-7721 cells was not observed (P<0.01). PTPS-I stimulated the proliferation of MNCs (P<0.01) and significantly enhanced the expression of TNF-α and IL-6 mRNA in MNCs (P<0.05, P<0.01). CONCLUSION:The results suggest that PTPS-I is an immunomodulator and its antitumoral activity is through the immunomodulatory mechanism rather than the direct cytotoxicity against tumor cells.     

Activated NK cells against human hepatocellular carcinoma cell lines in vitro and in vivo

AIM: To evaluate the role of natural killer(NK) cells against a variety of human hepatocellular carcinoma (HCC) cell lines in vitro and in vivo, and to investigate the expression of MHC class I chain-related protein (MIC protein) in these HCC cell lines. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 50 mL peripheral blood donored by a healthy volunteer and cultured in the NK cell kit followed by amplification and cytokine activation. The release of lactate dehydrogenase (LDH) was detected by lysis experiment. Animal experiments were performed following vaccination with HCC cell lines (BEL7402, HepG2 and SMMC7721) in BALB/c-nu/nu mice. The diameters of the tumors were measured and the growth curves of difference cell lines were delineated. Three weeks later, these mice were sacrificed and the weight of the transplanted tumors was also detected. Furthermore, the expression of MIC protein was determined. RESULTS: The obvious differences of lysis effects of NK cells on different cell lines were observed, in which the lysis effect of NK cells on K562 cells was the strongest, the effect on BEL7402 cells was in the middle and the effect on SMMC7721 cells was the weakest. In animal experiments, NK cells also showed obvious and different restraining effects on a variety of HCC cell line-transplanted tumors in nude mice. The tumor inhibitory rates of NK cells were 43.5% in BEL-7402 cell-transplanted tumor, 40.7% in HepG2 cell-transplanted tumor and 36.0% in SMMC-7721 cell-transplanted tumor. The protein expression of MIC was 48.7%, 32.8% and 0.9% in the 3 cell lines,respectively. CONCLUSION: The lysis effects of NK cells on a variety of human HCC cell lines are different. The expression of MIC in the tumor cells may play distinct role in evaluating these differences.