Expression of breast cancer resistance protein and p-glycoprotein in residual breast cancer tissue after chemotherapy and its correlation with cancer stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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) .</p><p><b>CONCLUSION</b>Chemotherapy endows residual breast cancer tissue with cancer stem cells-like features, leading to multidrug resistance of breast cancer.</p>

Inhibitory effect of pyrrolidine dithiocarbamate combined with matrine on the growth of human hepatocellular carcinoma xenografts / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>

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

To study the biological characteristics and resistant mechanisms of the cisplatin-resistant human hepatocellular carcinoma HCC cell line. 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. 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. 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

Inhibition of NF-kappa B activity enhanced apoptosis induced by matrine in hepatocellular carcinoma cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Effect of epigallocatechin-3-gallate on the invasiveness of hepatocarcinoma cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>The effect of EGCG on the invasion of HepG2 cells in vitro was studied.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>EGCG has anti-invasion effects on HepG2 cells.</p>

Separation and identification of the exosomes derived from a mouse hepatoma carcinoma cell line (H22) and initial investigation of their protein composition / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Study on the angiogenic ability of tumor fibroblasts / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To explore the role of fibroblasts derived from tumor in the tumor angiogenesis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>The results suggested that fibroblasts in tumors secrete more growth factors and angiogenic factors to promote the angiogenesis and invasion of solid tumors.</p>