Breast cancer stem cells cultured without insulin and effect of estrogen induction

AIM:To determine whether suspension culture medium without insulin can be used to feed breast cancer tumorsphere, or not. METHODS:MCF7 cells were used to build tumorsphere. The morphological changes, CD44+ CD24- expression, aldehyde dehydrogenase 1 (ALDH1) expression and multiple division ability were measured to identify the breast cancer stem cells and to detect the function of 17β-estradiol (E2β) in tumorsphere of MCF7 cells. RESULTS:The tumorshere, each containing 30 to 60 cells, was obtained by the method of insulin-removal suspension culture. These cells were cytokeratin 18 and CD10 proteins positive, and the number of CD44+ CD24- cells and ALDH1 protein expression were significantly higher than the adherent cultured cells (P<0.05). Using 10-10 mol/L E2β to treat the tumorshere for 7 d, the tumor cell number and volume were significantly increased. Using 10-10 mol/L E2β to treat the tumorshere for 24 h, the CD44+ CD24-cells and ALDH1 protein expression were significantly higher than those in non-treatment group (P<0.05). CONCLUSION: Suspension culture medium without insulin can be used to feed breast cancer tumorsphere. These tumorsphere could be used as a model to determine the function of E2β in breast cancer stem cell research.     

Expression and clinical significance of RhoC and Ki-67 in human esophageal squamous cell carcinoma tissues

AIM: To investigate the expression and significance of RhoC and Ki-67 in human esophageal squamous cell carcinoma (ESCC) tissues.METHODS: The expression of RhoC and Ki-67 was detected in 52 specimens of ESCC by the method of immunohistochemistry. The clinicopathological features were also analyzed.RESULTS: The expression of RhoC was detected in 32 of the total 52 (61.5%) cases of human ESCC tissues, significantly higher than that in the adjacent histologically normal epithelium, which was only in 11 of 37 cases (29.7%, P<0.05). RhoC expression was closely correlated with clinical tumor-node-metastasis (TNM) stage (P<0.05) and lymph node metastasis (P<0.05) in ESCC. The over-expression of RhoC was positively correlated with Ki-67 in ESCC (r=0.322, P<0.05).CONCLUSION: The over-expression of RhoC protein significantly correlates with advanced TNM stage, lymph node metastasis and cell proliferation ability of ESCC. Therefore, RhoC may be a new auxiliary parameter for early diagnosis and prognostic evaluation of ESCC.     

食管癌切除术后并发胸胃排空障碍的诊断与防治

从１９７３年１月至１９９５年２月治疗食管癌切除术后胸胃排空障碍１１例。１０例行１１次手术：膈下胸胃十二指肠吻合术１例，胸内胸胃空肠合术２例，幽门区纵行全层切开横行缝合术１例；粘连松解术７例，其中同时行十二指肠松解术１例，幽门成形术２例。术后均解除梗阻。还就此症的诊断和预防作了讨论。     

Endocrine differentiation and its mechanism in endometrial carcinoma

AIM:To investigate endocrine differentiation and its mechanism in endometrial carcinoma.METHODS:Endocrine cells (EC) were identified by immunohistochemical staining of chromogranin A in 50 cases of endometrioid adenocarcinoma and 20 cases of normal endometrium. Double-label techniques were used for simultaneous demonstration of the CgA and cytokeratin.RESULTS:The positive rate of CgA in endometrioid adenocarcinoma was 44.0%, and was significant higher than that in normal endometrium (15.0%, P＜0.05). Scanty EC was present in normal endometrium. The number and staining intensity of EC in endometrioid adenocarcinoma were greater than that in normal. Co-expression of CgA and cytokeratin were detected in endometrioid adenocarcinoma.CONCLUSION:The presence of endocrine cells in endometrioid adenocarcinoma showed heterogeneity of tumors. The occurrence of "Multidirectional differentiation cells" within the endometrioid adenocarcinoma may indicate that endocrine cells derive from malignant cells of multidirectional differentiation.     

Non-steroidal anti-inflammatory drugs induce apoptosis in implanted hepatoma of mice

AIM: To study the mechanism of the effect of NSAIDs on apoptosis in mice hepatoma at anti-inflammatory doses. METHODS: Kunming breed mice were inoculated subcutaneously in the flank with mice hepatoma H22 cell line. The effects of ibuprofen, indomethacin, and nimesulide on apoptosis were determined by using electron microscopy, agarose gel electrophoresis, flow cytometry, and Western blot analysis of the expression of c-myc, bcl-x_L and bcl-2 proteins. RESULTS: NSAIDs induced apoptosis of mice implanted hepatoma, which includes the morphological changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the "ladder pattern" revealed by agarose gel electrophoresis of DNA. The apoptotic index was increased to 15%±1.0%, 29.7%±1.5%, 46.3%±3.5% from 3.3%±0.6% by detecting Sub-G₁ peaks on flow cytometry. Western blot analysis showed that levels of bcl-2 and bcl-x_L were significantly reduced by treatment with nimesulide. Ibuprofen and indomethacin decreased bcl-2 expression but increased bcl-x_L expression. C-myc wasn't changed in these groups. CONCLUSION: These results suggest that NSAIDs induces apoptosis of mice hepatoma, which may be due to their regulation on the expressions of bcl-2 family genes.     

Relationship between expression of MKK-4 and MMP-9 genes in primary hepatic carcinoma with or without invasion and metastasis

AIM：To investigate the expression of mitogen activated protein kinase kinase 4 (MKK-4) and MMP-9 mRNA in primary hepatic carcinoma (PHC), and to analyze its relationship with invasion and metastasis. METHODS：The expression of MKK-4 and MMP-9 mRNA in 34 cases of hepatic carcinoma tissues and adjacent tissues,and in 12 cases of normal liver tissues were detected with RT-PCR. RESULTS：(1) The expression level of MMP-9 mRNA was higher in metastatic cancer tissues than that in other tissuses (P<0.01). (2) There was significant statistical difference among the expression level of MKK-4 mRNA, but the level in metastatic cancer was low (P<0.01). (3) There was no statistical difference among the expression level of MKK-4 or MMP-9 mRNA among the adjacent tissues and normal tissues (P>0.05). (4) MMP-9 mRNA had a tendency to rise as PHC became invasive and metastatic.The expression level of MKK-4 had a tendency to decline in PHC became invasive and metastatic. (5) The expression level of MMP-9 or MKK-4 mRNA had no correlations with tumor volume,or cell differentiation (P>0.05). (6) There were correlations between expressions of MKK-4 and MMP-9 mRNA in PHC (Pearson Correlation, r=-0.925, P<0.01). CONCLUSION：There are high MMP-9 mRNA expression and low MKK-4 mRNA expression in PHC.The expression level of MKK-4 or MMP-9 mRNA is correlated with tumor metastasis.     

The latent gene expression of Epstein-Barr virus in nasopharyngeal carcinoma

AIM: To examine the latent membrane protein 1(LMP1)-DNA sequence in nasopharyngeal carcinoma(NPC) and detect mRNA expression of LMP1,EBNA1,EBNA2,and to explore the relationship between EBV infectious status,expression products and NPC carcinogenesis.METHODS: LMP1 DNA was detected in NPC by PCR.Direct sequence was applied to analyze the difference between NPC-LMP1-DNA and B95-8- LMP1-DNA.mRNA expressions of LMP1,EBNA1,EBNA2 in NPC were detected by nested RT-PCR.RESULTS: LMP1 DNA existed in all 47 NPC tissues.Several single nucleotide variations were found between NPC-LMP1-DNA and B95-8- LMP1-DNA.The notable variation was the lost of XhoⅠrestriction site in NPC.Direct sequence showed 30 bp deletion in NPC.The mRNA expressions of LMP1,EBNA1 and EBNA2 in NPC were 76.6%,80.0% and 74.5% respectively by nested RT-PCR.The expression of EBNA1 in NPC was promoted by Q promoter while the expression of EBNA1 in B95-8 was promoted by C promoter.CONCLUSION: The way of EBV involved in NPC is complex.Latent genes such as LMP1,EBNA1 and EBNA2 as well as early lytic gene BARF1 may all play certain roles in NPC carcinogenesis.     

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.