The anticancer activity of genistein on implanted tumor of human primary gastric carcinoma cells in nude mice

AIM: To investigate the apoptosis of implanted tumor of primary human gastric cancer cells in nude mice induced by genistein and the relation between this apoptosis and expression of bcl-2 and bax.METHODS: Establishing a transplanted tumor model by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice.The different doses of genistein (0.5mg/kg,1mg/kg and 1.5 mg/kg ) were directly injected beside tumor body respectively,for six times at an interval of two days.Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated.We observed the morphologic alteration by electron microscope,measured the apoptotic rate by TUNEL staining method,detected the expression of apoptosis-regulated gene bcl-2 and bax by immunohistochemical staining and RT-PCR.RESULTS: Genistein could significantly inhibit carcinoma growth when it was injected near the carcinoma.Genistein induced implanted tumors cells to undergo apoptosis with apoptotic characteristics by transmission electron microscope.The apoptosis index of above three groups was increased progressively.Positive rate of Bcl-2 protein of above three groups was decreased progressively and positive rate of Bax protein of above three groups was increased progressively by immunohistochemical staining.The density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR.CONCLUSION: Genistein is able to induce the apoptosis of transplanted tumor cells.This apoptosis may be mediated by down-regulating bcl-2 and up-regulating bax mRNA and its protein.     

Effect of lung cancer cells transfected with interleukin 18 gene on the immunological activity of dendritic cells

AIM: To study the effect of interleukin 18 gene transfected lung cancer cells on the phenotype and immunological activity of dendritic cells (DC). METHODS: A secretive IL-18 expression vector containing IL-12 P40 signal sequence was constructed and transfected into NCI-H460 lung cancer cells. DC induced from human peripheral blood were divided into 4 groups (NT, PV, GT and PD). DC were stimulated by non-transfected NCI-H460 cells, pure vector transfected NCI-H460 cells and IL-18 transfected NCI-H460 cells respectively for group NT, PV, GT, and non-stimulated DC for group PD. CD54, CD80, CD83 and CD86 on DC in the 4 groups were detected with flow cytometry. T cell proliferation stimulated by DC in the 4 groups was assayed with MTT method. IL-12 release in cultured DC supernatant was measured by ELISA. RESULTS: Sequencing result of the secretive IL-18 was correct. The transfected cells expressed IL-18 fusion gene and 18 kD IL-18 protein. DC in GT group expressed more surface molecules than those in other 3 groups. T cell proliferation and IL-12 secretion in GT group were higher than those in other 3 groups. CONCLUSION: IL-18 gene transfected NCI-H460 cell increases surface molecule expressions on DC. It also enhances immunological activity and IL-12 secretion in DC.     

Construction and identification of eukaryotic expression vector of bcl-2 siRNA

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.     

Antiproliferation effects of 188Re labeled monoclonal antibody on prostate cancer cells in vitro

AIM: To investigate the effect of 188Re labeled monoclonal antibody on prostatic specific membrane antigen 7E11C5.3,radioimmunotherapy for the treatment of human prostate cancer cell line LNCaP in vitro.METHODS: 188Re-7E11C5.3 was prepared by direct 2-mercaptoethanol reduction method.Labeling efficiency and radiochemical purity was measured by paper chromatography.Immunoreactive fraction was determined by linear extrapolation.Cytotoxicity to LNCaP cells was determined by MTT assay.RESULTS: The labeling yield of 188Re-7E11C5.3 was (93.16±2.18)%,the radiochemical purity was (95.62±0.48)%,and the immunoreactive fraction was (74.86±1.86)%.The inhibitory effect of 188Re-7E11C5.3 on cell proliferation of LNCaP cells was significantly higher than that of 188Re-mIgG or 188ReO-4.The 50% inhibitory doses (IC50) of 188Re-7E11C5.3,188Re-mIgG,and 188ReO-4 were (23.38±3.73)×107 Bq/L,(59.21±8.02)×107 Bq/L and (68.89±10.91)×107 Bq/L,respectively.CONCLUSION: 188Re-7E11C5.3 can effectively inhibit the growth of in vitro cultured prostate cancer cells and shows much potential for prostate cancer radioimmunotherapy.     

Nucleostemin gene expression in human breast tumor tissues and its relation with clinical pathology

AIM: To study the expression of nucleostemin (NS) gene in human breast tumor tissues and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor.METHODS: Total RNA was isolated from human breast tumor tissue.The methods of electrophoresis and RT-PCR were used in measuring NS gene expression level,and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor were analyzed.RESULTS: The results indicated that there was no NS gene expression detected in normal breast tissues,and NS gene expression in malignant breast tumor tissues (P<0.01) was higher than that in the benign breast tumor tissues.The higher histological grades of the breast cancer showed the stronger NS gene expression (P<0.01),the higher TNM stages of the breast cancer showed the stronger NS gene expression (P<0.01),and the level of NS gene expression had not correlation with the histological types (P>0.05).CONCLUSION: It is suggested that there is no relation of NS gene expression level with histological types of the breast cancer,but there is a marked correlation of NS gene expression level with the histological grades and TNM stages.     

Effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene in DLD1 colon cancer cells

AIM:To evaluate effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene and clarify the involved mechanisms in DLD1-TRAIL/R colon cancer cells. METHODS:Human colon cancer cell line DLD1-TRAIL/R cells that were resistant to TRAIL-expressing adenovector (Ad/gTRAIL) were treated with Ad/gTRAIL combined with different chemotherapeutic agents. Then, the cell viability was measured by MTT method, and apoptotic signaling conditions, including activation of caspase-3 and caspase-8, expression of Bax and Bcl-XL, were measured by Western blotting analysis. RESULTS:In vitro data showed that several chemotherapeutic agents, including 5-fluorouracil (5-FU) and mitomycin c (MMC), overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R colon cancer cells. The combination of Ad/gTRAIL and 5-FU effectively suppressed tumor growth in vivo in subcutaneous tumors established from DLD1-TRAIL/R cells. Further data showed that treatment with the combination of Ad/gTRAIL and 5-FU or MMC led to enhance the activation of caspase-3. Moreover, MMC but not 5-FU induced overexpression of Bax gene that was sufficient to overcome the resistance to TRAIL gene in DLD1-TRAIL/R cells. CONCLUSION:Chemotherapeutic agents, such as 5-FU and MMC, overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R cells. The candidate mechanisms for MMC but not 5-FU to overcome this resistance might involve the induction of over-expressed Bax protein in DLD1-TRAIL/R cells.     

Regulation of estrogen on the expression of KLK6 mRNA and protein in human breast cancer cell line MCF-7

AIM:To investigate the effects of estrogen and tamoxifen on the expression of KLK6 mRNA and protein (hK6) in human breast cancer cell line MCF-7. METHODS:MCF-7 cells were incubated with 17-βE2 and tamoxifen at different concentrations for 72 hours, respectively. The expression levels of kallikrein 6 (KLK6) mRNA and protein were evaluated by fluorescence quantitative RT-PCR and flow cytometry, respectively. RESULTS:Compared with ethanol control, KLK6 mRNA expression levels were significantly decreased when 17-βE2 was added at concentrations of 10^-10 and 10^-8 mol/L (P<0.01). No statistical change was observed when 17-βE2 was at 10^-12 mol/L (P>0.05). Flow cytometry showed the same results. The average fluorescence intensity (AFI) that represents the level of hK6 was decreased after incubated with 17-βE2 (P<0.01). After incubation with tamoxifen, the levels of KLK6 mRNA and hK6 were increased (P<0.01). CONCLUSION:Estrogen down-regulates the expression levels of KLK6 mRNA and protein (hK6), while tamoxifen has an opposite effect.