[Silencing hypoxia inducible factor-2α gene by small interference RNA inhibits the growth of mammosphere cells in nude mice under hypoxic microenvironment].

OBJECTIVE: To explore the effects of silencing hypoxia inducible factor-2α (HIF-2α) by small interference RNA on the growth of mammosphere cells in nude mice under hypoxic microenvironment. METHODS: The empty and interference vectors were transfected into MCF-7 cell. Then G418 was added to screen the positive cells to obtain stable cell line. The empty and interference vectors were inoculated subcutaneously into left and right back near hind limb of nude mice. The volume and weight of tumors were calculated respectively. The expressions of HIF-2α, CD44, OCT-4 and KLF-4 protein in xenograft tumor tissues were detected by Western blot. RESULTS: The expression vector of HIF-2α-siRNA was successfully established. The formation of mammosphere was lowered by silencing HIF-2α gene expression. In contract to empty vector group cell, there were obvious decreases in the volumes and weights of tumors in interference group (P < 0.05). The expression of HIF-2α protein of interference group (0.42 ± 0.01) was much lower than that of the empty vector group (0.89 ± 0.03, P < 0.05), the expression of CD44 protein of interference group (0.21 ± 0.01) was much lower than the empty vector group (0.78 ± 0.03, P < 0.05), the expression of OCT-4 protein of interference group (0.42 ± 0.01)was much lower than the empty vector group (0.68 ± 0.03, P < 0.05) and the expression of KLF-4 protein of interference group (0.34 ± 0.01) was much lower than the empty vector group (0.72 ± 0.03, P < 0.05). CONCLUSION: Silencing HIF-2α gene can effectively inhibit the growth of breast cancer stem cells in nude mice under hypoxic microenvironment. Its mechanism may be through inhibited capacity for self-renewal and proliferation of breast cancer stem cells in vivo through the down-regulated expressions of markers associated with stem cells. HIF-2α is expected to become a new target for gene therapy of breast cancer.

Expression of DNMTs and genomic DNA methylation in gastric signet ring cell carcinoma.

The aim of the present study was to investigate the protein expression of DNA methyltransferases (DNMTs) and genomic DNA methylation status of genomes in gastric signet ring cell carcinoma (SRC). Immunohistochemistry was performed to analyze DNMT expression and methylated DNA immunoprecipitation microarray (MeDIP­chip) and MeDIP quantitative real­time PCR (MeDIP­qPCR) were performed to analyze the genomic DNA methylation status in gastric SRC tissue. An increase in DNMT1 and decrease in DNMT3A expression in SRC tissue was observed compared with matched non­cancerous tissue. However, expression of other DNMTs, DNMT2, DNMT3B and DNMT3L, was not found to differ significantly between carcinoma and control. The MeDIP­chip assay revealed that methylation of gene promoters and CpG islands in SRC was higher than those in matched control tissue. However, MeDIP­qPCR analysis demonstrated that specific tumor­related genes, including ABL2, FGF18, TRAF2, EGFL7 and RAB33A were aberrantly hypomethylated in SRC tissue. Results of the current study indicate that gastric SRC may produce complex patterns of aberrant DNA methylation and DNMT expression.

[Expression of breast cancer resistance protein and p-glycoprotein in residual breast cancer tissue after chemotherapy and its correlation with cancer stem cells].

OBJECTIVE: To compare the expression differences of breast cancer resistance protein(BCRP/ABCG2) and P-glycoprotein(P-gp) in breast cancer tissue before chemotherapy and in residual breast cancer tissue, and to explore its correlation with breast cancer stem cells. METHODS: Immunohistochemistry was used to detect the expression of ABCG2, P-gp, and breast cancer stem cells(BCSCs) markers(CD44 and CD24) in breast cancer tissue before chemotherapy and residual breast cancer tissue after chemotherapy. Immunofluorescence was applied for determination of the CD44 and CD24 protein expressions of BCSCs microspheres cells. The monoclone-forming ability of BCSCs microspheres cells was detected by limited dilution assay. The expressions of ABCG2, P-gp, CD44, and CD24 proteins were detected by Western blot. RESULTS: Compared with those in breast cancer tissue before chemotherapy, the expression levels of ABCG2 and P-gp were positively correlated with the expression level of CD44 protein(Χ(2)=41.34, r=0.83;Χ(2)=22.81, r=0.61) in residual breast cancer tissue after chemotherapy;meanwhile, they were negatively correlated with the expression of CD24 protein(Χ(2)=-21.25, r=0.72;Χ(2)=-17.26, r=0.65) (all P<0.05) .The diameter of BCSCs microspheres were increased significantly after chemotherapy.The content of BCSCs increased by about 2.5 times after chemotherapy.The expressions of ABCG2, P-gp and CD44 proteins significantly increased and that of CD24 protein significantly declined(P<0.05) . CONCLUSION: Chemotherapy endows residual breast cancer tissue with cancer stem cells-like features, leading to multidrug resistance of breast cancer.

Expression of DNMTs and MBD2 in GIST.

The aim of this study was to investigate the protein expression of DNA methyltransferases (DNMTs, including DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L) and methyl-CpG-binding domain protein 2 (MBD2) in gastrointestinal stromal tumor (GIST). Immunohistochemistry and western blot analysis were used to detect expression of DNMT and MBD2 in 15 pairs of adult GIST and matched non-tumor tissues. The protein expression of DNMT1, DNMT2, DNMT3B, DNMT3L and MBD2 was significantly higher in adult GISTs compared to the matched non-tumor tissues (P<0.05). However, no significant difference was detected in the protein expression of DNMT3A between tumor and non-tumor tissues (P>0.05). Associations between DNMT1 expression and mitotic index, DNMT3B expression and tumor size, as well as DNMT3L expression and Helicobacter pylori infection were detected in GISTs (P<0.05). In conclusion, GISTs exhibit a high protein expression of DNMT (with the exception of DNMT3A) and MBD2.

Integrin-linked kinase functions as a tumor promoter in bladder transitional cell carcinoma.

The aim of this study was to elucidate the role of the integrin-linked kinase (ILK) gene in development of human bladder transitional cell carcinoma (BTCC). Expression of ILK protein and ILK mRNA in 56 cases of human BTCC tissue and in 30 cases of adjacent normal bladder tissue was detected by immunohistochemistry S-P and reverse transcription polymerase chain reaction (RT-PCR), respectively. Four specific miRNA RNAi vectors targeting human ILK were synthesized and transfected into BIU-87 cells by liposome to obtain stable expression cell strains. The influence of ILK on proliferation of BTCC was detected by MTT, FCM on athymic mouse tumorigenesis. The positive rate of ILK protein in BTCC tissue (53.6%) was much higher than adjacent normal bladder tissue (10.0%) (p<0.05). Similarly, expression of ILK mRNA in BTCC tissue (0.540 ± 0.083) was significantly higher than in adjacent normal bladder tissue (0.492 ± 0.070) (p<0.05). MTT showed that the proliferation ability of miRNA-ILK transfected group was clearly decreased (p<0.05), the cell cycle being arrested in G0/G1-S, an tumorigenesis in vivo was also significantly reduced (p<0.05). ILK gene transcription and protein expression may be involved in the development of BTCC, so that ILK might be the new marker for early diagnosis and the new target for gene treatment.

[Inhibitory effect of pyrrolidine dithiocarbamate combined with matrine on the growth of human hepatocellular carcinoma xenografts].

OBJECTIVE: To investigate the relationship between activation of nuclear factor-K-gene binding (NF-κB) and apoptosis induced by matrine(MT) in transplanted tumor of human hepatocellular carcinoma in nude mouse. METHODS: Tumors were established by injection of hepatocellular carcinoma cell line HepG2 into the back of nude mice. The mice were divided randomly into four groups: Control group, MT group (35 mg/kg), PDTC group (120 mg/kg) and Combination group: PDTC + MT group (120 mg/kg + 35 mg/kg), the reagents were injected peritoneally. The tumor growth curve of nude mice bearing transplanted tumor were observed and the inhibition ratios were evaluated. Apoptosis of carcinoma cells was analyzed by TUNEL. The DNA-binding activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Expression of bcl-2 and bax in carcinoma tissue were detected by immunohistochemical method. NF-κB mRNA, bcl-2 mRNA and bax mRNA in carcinoma tissue were detected by RT-PCR. RESULTS: Pyrrolidine dithiocarbamate (PDTC) could enhance the inhibition of matrine on carcinoma proliferation (P < 0.05). The apoptosis and activation of NF-κB in carcinoma cells could be induced by matrine. PDTC significantly suppressed NF-κB activation induced by matrine in carcinoma cells from 93.64 ± 2.95 to 65.78 ± 5.65 (F = 124.754, P < 0.01). Meanwhile, PDTC increased the apoptosis induced by matrine from 55.9% ± 2.8% to 74.3% ± 4.8% (P < 0.05).A positive correlation observed between the expressions of NF-κB and of bcl-2 (Pearson correlation coefficient = 0.983, P < 0.01). CONCLUSIONS: Matrine could induce apoptosis and activation of NF-κB in transplanted tumor. PDTC could increase apoptosis in hepatocellular carcinoma cells might be due to the suppression of NF-κB activation and the enhancement of bcl-2 expression.

Overexpression of Forkhead Box M1 transcription factor and nuclear factor-κB in laryngeal squamous cell carcinoma: a potential indicator for poor prognosis.

The Forkhead Box M1 transcription factor and nuclear factor-κB have been shown to play important roles in the development and progression of human cancers. However, the functional significance of Forkhead Box M1 transcription factor in laryngeal squamous cell carcinoma and the correlation between Forkhead Box M1 transcription factor and nuclear factor-κB remain unclear. In the current study, we have shown that Forkhead Box M1 transcription factor and nuclear factor-κB were significantly overexpressed in laryngeal squamous cell carcinoma tissues and precancerous lesions, compared with adjacent normal tissues (both P < .001). The overexpression of Forkhead Box M1 transcription factor was significantly associated with histologic differentiation (rs = 0.321, P = .002), T stage (rs = 0.276, P = .009), lymph node metastasis (rs = 0.266, P = .012), and clinical stage (rs = 0.272, P = .010); overexpression of nuclear factor-κB was significantly associated with T stage (rs = 0.404, P < .001), lymph node metastasis (rs = 0.293, P = .005), and clinical stage (rs = 0.425, P < .001). Overexpressions of both Forkhead Box M1 transcription factor and nuclear factor-κB were associated with worse overall survival (P = .041 and P < .001, respectively). Multivariate Cox regression analysis showed that T stage, lymph node metastasis, and nuclear factor-κB were independent prognostic factors for laryngeal squamous cell carcinoma (P = .038, P = .014, and P = .005, respectively). Furthermore, a significant correlation was observed between Forkhead Box M1 transcription factor and nuclear factor-κB (rs = 0.683, P < .001), indicating the potential direct or indirect interaction between them. In conclusion, our results suggest that overexpressions of Forkhead Box M1 transcription factor and nuclear factor-κB and the possible interaction between them may play important roles in the development and progression of laryngeal squamous cell carcinoma, and Forkhead Box M1 transcription factor and nuclear factor-κB may serve as useful prognostic markers for laryngeal squamous cell carcinoma.

[Expression of paxillin in breast cancer cell with high and low metastatic potentiality].

OBJECTIVE: To study the expression of paxillin in the two subgroups of human breast cancer cells with high and low metastatic potentialities and the effect of paxillin on tumor metastasis and adhesion. METHODS: Human breast cancer MDA-MB-435s cells were cultured in Transwell chamber with artificial matrigel, two subgroups of MDA-MB-435s cells with different metastatic potentiality were obtained by the ability of cells penetrating artificial matrigel. The expressions of paxillin mRNA and protein were examined by Immune histochemistry, Western blot and RT-PCR. The adhesion rates of the cells were examined by MTT. RESULTS: It was revealed by Immune histochemistry, Western blot, and RT-PCR that the expressions of paxillin at mRNA and protein levels were significantly higher in the cells with high metastatic potentiality. The difference was significant (t = 25.02,10.10, 17.18, P < 0.01). The adhesion rate was higher in the cells with high metastatic potentiality, and the difference was significant (F = 41.87, P < 0.01). CONCLUSION: The expression of paxillin may play an important role in human breast cancer metastasis and adhesion.

The role of Crk/Dock180/Rac1 pathway in the malignant behavior of human ovarian cancer cell SKOV3.

Small GTPases, particularly the Rho family, are key regulators of cell motility and migration. Dock180 was well known for the main target of signal adaptor protein Crk and acted as a guanine-nucleotide exchange factor for small GTPase Rac1. In the present study, Dock180 was found to combine primarily with CrkI other than CrkII, and its association with Elmo1 was also demonstrated in ovarian cancer cell SKOV3. To evaluate the role of Dock180 in human ovarian cancer cell, we performed RNAi-mediated knockdown of Dock180 in SKOV3 cells using small interfering RNA expression vector. In Dock180 knockdown cells, we found that Elmo1 expression and Rac1 activity were decreased simultaneously. By contrast, the expressions of both another Crk-combining molecule C3G and Rap1 activity were observed to increase obviously. Accordingly, all Dock180 knockdown cells present with evident change in cell morphology, reduced cell proliferation, and attenuated cell migration. Taken together, these results suggest that signal transfer of Crk/Dock180/Rac1 is implicated in actin cytoskeleton reorganization and thus in the cell proliferation, motility, invasion, and of human ovarian cancer cell line SKOV3.

Characterization and resistance mechanisms of cisplatin-resistant human hepatocellular carcinoma cell line.

OBJECTIVE: To study the biological characteristics and resistant mechanisms of the cisplatin-resistant human hepatocellular carcinoma (HCC) cell line. METHODS: The study took place in the Department of Pharmacology, Chongqing Medical University, Chongqing, China, between April 2005 and November 2007. A resistant HCC cell line (QGY/CDDP) was established by stepwise increasing cisplatin (CDDP) concentration and intermittent administration. Drug-chemo sensitivity was detected by 3-4,5-dimethylthiazol-2yl-2,5- diphenyltetrazolium bromide (MTT) assay. Cell doubling time was determined by cell counting, and cell cycle analysis was performed by flow cytometric (FCM) assay. Intracellular platinum accumulation was detected by atomic absorption spectrometry and the expression of P-glycoprotein (P-gp) and glutathione S-transferase-pi (GST-pi) were analyzed by FCM assay. RESULTS: QGY/CDDP cell line was established after 3 months with stable resistance to CDDP and exhibited cross-resistance to many other chemotherapeutic agents. Compared with parental cell line, cell doubling time of QGY/CDDP prolonged; and the cell proportion decreased in S and G2/M-phase and increased in G0/G1-phase. In QGY/CDDP cells, intracellular platinum accumulation decreased and GST-pi expression increased, but P-gp expression kept stable. CONCLUSION: QGY/CDDP cell line shows the typical and stable resistant phenotype and characteristics of resistant cells. Its mechanisms of resistance to CDDP may be mediated by reduced accumulation of intracellular platinum and higher GST-pi expression, but it is not associated with P-gp expression.

[Separation and identification of the exosomes derived from a mouse hepatoma carcinoma cell line (H22) and initial investigation of their protein composition].

OBJECTIVE: To separate and identify the exosomes derived from a mouse hepatoma carcinoma cell line (H22) and to detect their protein composition, and to investigate the possibility of using these exosomes as a kind of tumor vaccine. METHODS: Exosomes were purified by serial ultracentrifugation and sugar density ultracentrifugation, and then they were observed and identified by electron microscopy. Exosomes underwent peptide mass fingerprint and Western blot analyses. RESULTS: H22 cell-derived exosomes were 20-90 nm round or oval vesicles. The exosomes expressed HSP70, ICAM-1, EF-G2, DLC-A, C-myc protein and Vav-2 protein. CONCLUSION: Serial ultracentrifugation and sugar density ultracentrifugation can be used to purify H22 cell-derived exosomes. H22 cell-derived exosomes express a distinct set of proteins involving in and/or relating to antigen presentation (HSP70, ICAM-1), migration (DLC-A), adhesion (ICAM-1), cytoskeleton (EF-G2) and tumour antigens (C-myc, Vav-2). The exosomes have immunogenicity.

[Effect of epigallocatechin-3-gallate on the invasiveness of hepatocarcinoma cells in vitro].

OBJECTIVE: The effect of EGCG on the invasion of HepG2 cells in vitro was studied. METHODS: The expressions of MUC1 in HepG2 cells before and after they were treated with EGCG were studied with immunohistochemistry. The effects of EGCG on the invasion of HepG2 cells were evaluated using Transwell chambers attached with polycarbonate filters and reconstituted basement membranes (Matrigel). Gelatin zymography was performed to detect the secretion of MMP-2 and MMP-9. RESULTS: EGCG reduced the expression of MUC1, suppressed significantly the invasion of tumor cells into the basement membranes (P < 0.01) and reduced the secretion of MMP-2, MMP-9. CONCLUSION: EGCG has anti-invasion effects on HepG2 cells.

[Inhibition of NF-kappa B activity enhanced apoptosis induced by matrine in hepatocellular carcinoma cells].

OBJECTIVE: To investigate the relationship between activation of nuclear factor-kappa gene binding (NF-kappaB) and apoptosis induced by matrine in hepatocellular carcinoma cell line HepG2. METHODS: HepG2 cells were stimulated by different concentrations of matrine (0.8, 1.0, 1.5, 2.0, 2.5 g/L). The HepG2 cell survival rates were evaluated by MTT assay. Cultured HepG2 cells were implanted in culture flasks and divided into four groups: a control group, a pyrrolidine dithiocarbamate (PDTC) group (20 micromol/L), a matrine group (1.5 g/L) and a combination group, PDTC (20 micromol/L) + matrine (1.5 g/L) combination group. Apoptosis induced by matrine was analyzed by flow cytometry (FCM) and TUNEL. The DNA-binding activity of NF-kappaB was determined by electrophoretic mobility shift assay (EMSA). RESULTS: PDTC enhanced the inhibition of matrine on cell proliferation (F=183.92, P less than 0.01). The apoptosis and activation of NF-kappaB of HepG2 cells were induced by matrine. PDTC significantly suppressed NF-kappaB activation induced by matrine in HepG2 cells. PDTC increased the apoptosis induced by matrine of the HepG2 cells from 6.11% +/- 0.81% to 12.95% +/- 0.02%, chi2=9.67, P less than 0.01. CONCLUSIONS: Matrine could induce apoptosis, and at the same time induce activation of NF-kappaB in HepG2 cells. PDTC increases the apoptosis in hepatocellular carcinoma cells and it may be related to suppressing NF-kB activation of HepG2 cells.

[Effects of TNBG on the proliferation and apoptosis of human hepatocellular carcinoma cell line QGY-7701].

OBJECTIVE: To investigate the effects and mechanism of TNBG on the proliferation and apoptosis of human hepatocellular carcinoma cell line QGY-7701. METHODS: The 3H-TdR incorporation and MTT assay were used to test the effects of anti-proliferation and cytotoxicity respectively, the morphological changes of the cancer cells were examined under light and electron microscopes. The Flow cytometric analysis was performed to detect the cell cycle distribution and apoptosis. RESULTS: Marked anti- proliferation effect was observed after intervention of 2 microg/ml TNBG for 24 hours,and TNBG killed the cells in concentrition- and time-dependent manners. A lot of lipid droplets accumulated at the cytoplasm under light microscopy, and were confirmed by electron microscopy. Furthermore,the cells showed G2/M arrest and apoptosis. CONCLUSION: The above findings indicate that TNBG inhibits proliferation of tumor cells, induces apoptosis of tumor cells, and thus produces its antitumor effect.

[Study on the angiogenic ability of tumor fibroblasts].

OBJECTIVES: To explore the role of fibroblasts derived from tumor in the tumor angiogenesis. METHODS: Two-well co-culture system were used to detect the expression of MMP-9, TGF-beta1, TN and bcl-2 in L929-H22 cells, and their ability of promoting angiogenesis of ECV304 cells and invasion of MDA-MB-231 cells respectively, which were established in our laboratory before. Then their adhesion and the effect of their supernatant on H22 cells proliferation were analysed. RESULTS: Compared with L929 cells, the adhesion potential of L929-H22 cells increased (F>or=104.32, P<0.001), with the higher level of expression of MMP-9, bcl-2, TN, and TGF-beta1 in L929-H22 cells in creased (t>or=3.3055, P<0.01). L929-H22 and L929 cells enhanced the invasiveness of human mammary cancer MDA-MB-231 cells through artificial basement membrane (Matrigel) 1.21 and 0.48 times respectively (F=266.3, P<0.001). L929-H22 cells induced morphogenesis of ECV304 cells. L929-H22 stimulated endothelial cells to form more and longer tubes than L929 did (F>or=23.75, P<0.01). 25% CM of L929-H22 cells stimulated the growth of H22 cells (F=266.30, P<0.05). CONCLUSION: The results suggested that fibroblasts in tumors secrete more growth factors and angiogenic factors to promote the angiogenesis and invasion of solid tumors.

[Establishment of a cisplatin-induced human hepatocellular carcinoma drug-resistant cell line and its biological characteristics].

BACKGROUND & OBJECTIVE: Drug resistance is a popular topic in tumor study. The drug-resistant cell line established in vitro is generally used as the research model. To explore the mechanism of resistance of hepatocellular carcinoma(HCC) to cisplatin, we designed this study to establish a cisplatin-induced human hepatocellular carcinoma drug-resistant cell line and study its characteristics. METHODS: A resistant HCC cell line (QGY/cDDP) was established gradually by increasing dose of cisplatin and intermittent administration; drug sensitivity was detected by MTT assay; the changes of its biological characteristics were determined using light microscopy, electron microscopy, cell counting, flow cytometry(FCM), and chromatosome analysis. RESULTS: QGY/cDDP cell line was developed after 3 months with stable resistance to cisplatin and the resistance index was 10.81; QGY/cDDP cells exhibited cross-resistance to many other chemotherapeutic agents (5-Fuorouracil, epiadriamycin, etc). The morphology and chromatosome number of QGY/cDDP changed; doubling time prolonged; and the cell number of S-phase and G2/M-phase decreased while in G0/G1 phase increased compared with parent cells. CONCLUSION: QGY/cDDP cell line shows the typical and stable resistant phenotype.

Interaction between cisplatin, 5-fluorouracil and vincristine on human hepatoma cell line (7721).

AIM:To evaluate the killing effects of CDDP, 5-Fu and VCR on human hepaoma cell line (7721).METHODS:The median-effect principle was used.RESULTS:Killing effects of the individual drug were enhanced as the median concentration increased. Antagonism was produced when two drugs were used at a higher concentration (CI > 1), and synergism was achiened when CI < 1. Finally, the effect was influenced by both the ratios of drug concentration and the sequence of administration.CONCLUSION:The drug administration order and drug concentrations are significant factors that need to be considered in clinical practice.