Establishment and gene expression analysis of drug-resistant cell lines in hepatocellular carcinoma induced by sorafenib / 北京大学学报(医学版)

OBJECTIVE@#To establish the drug-resistant cell lines of hepatocellular carcinoma (HCC) induced by sorafenib, and to screen out the high expression genes in drug-resistant cell lines of HCC induced by sorafenib, then to explore the genes related to sorafenib resistance in hepatocellular carcinoma.@*METHODS@#The human PLC and Huh7 cell lines were obtained, then the PLC and Huh7 drug-resistant cell lines were induced with sorafenib by using intermittent induction in vitro. CCK8 assay was used to detect the IC50 value of sorafenib for evaluation of drug sensitivity of hepatocellular carcinoma cell lines in PLC and Huh7. All the up regulated genes in PLC and Huh7 drug-resistant cell lines induced by sorafenib were screened out using high-throughput cDNA sequencing (RNA-Seq), Ualcan database was used to analyze the correlations between the up regulated genes in PLC and Huh7 drug-resistant cell lines induced and four clinical biological characteristics of hepatocellular carcinoma, including the gene expressions between normal samples and tumor samples, tumor stage, tumor grade, and patient overall survival, to find the genes that might be involved in the mechanism of sorafenib resistance of hepatocellular carcinoma.@*RESULTS@#All the up regulated genes detected by the using high-throughput cDNA sequencing (RNA-Seq) in PLC and Huh7 drug-resistant cell lines were further screened out by following conditions:(1) genes co-expressed in PLC and Huh7 drug-resistant cells induced by sorafenib, (2) the fold change was more than 4 times and the difference was statistically significant (P <0.05), the top 12 up regulated genes in PLC and Huh7 drug-resistant cell lines were found, which were TPSG1, CBX4, CLC, CLEC18C, LGI4, F2RL1, S100A6, HABP2, C15ORF48, ZG16, FOLH1, and EPCAM. Compared with the correlations between the twelve genes and the clinical biological characteristics by Ualcan database, the potentially significant gene CBX4 was screened out.@*CONCLUSION@#The human PLC and Huh7 drug-resistant cell lines of hepatocellular carcinoma induced by sorafenib were successfully established. CBX4, the gene related to sorafenib resistance in hepatocellular carcinoma, was screened out by the high-throughput cDNA sequencing (RNA-Seq) and further analysis using Ualcan database, which is providing a powerful basis for further research on the mechanism of sorafenib resistance of hepatocellular carcinoma.

Experimental study on suppressive effect of zilongjin on growth of human breast cancer cell line MCF-7 in vitro and in vivo / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the suppressive effect and mechanism of action of Zilongjin (ZLJ, a composite Chinese drug) on the growth of human breast carcinoma cell line MCF-7 in vivo and in vivo.</p><p><b>METHODS</b>The cell survival rate and clone forming efficiency were observed by direct cell counting with trypan blue staining and double layers soft agar test; the p-ERK 1/2 expression was analyzed using Western blotting; 17-beta-estrogen pellet embedding was adopted to make the subcutaneous transplanted tumor MCF-7 cell in BALB/c nude mice for detecting the tumor growth suppressive effect of ZLJ (20 g crude drug/kg).</p><p><b>RESULTS</b>The survival rate of MCF-7 cell was obviously decreased by ZLJ in a time and dose-dependent manner; only few and small clones on soft agar could be found after treated with ZLJ, the inhibition rates of clone formation(%) for 0.75, 1.5, 3, 6 mg/mL of ZLJ were 12.66 +/- 1.54, 88.83 +/- 2.13, 100 and 100, respectively, as compared with that of non-treated. The expression of p-ERK 1/2 was suppressed and the ability of the tumorigenicity in nude mice was reduced effectively by ZLJ. The mean volumes and weights of tumor in the test group and the control group were (0.73 +/- 0.58) cm3 vs (1.36 +/- 0.64) cm3 and (1.02 +/- 0.25) g vs (1.66 +/- 0.09) g respectively, showing significant difference (P<0.05), and the tumor inhibition rate of ZLJ was 38.55%.</p><p><b>CONCLUSION</b>ZLJ shows obviously suppressive actions on malignant proliferation, transformation and tumorigenicity of human breast carcinoma cell line MCF-7; the down-regulation of p-ERK 1/2 protein may involve in these effects.</p>

Effects of zilongjin on proliferation and apoptosis of human breast cancer cell line MCF-7 / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Zilongjin (ZLJ, a composite Chinese drug) on proliferation and apoptosis of human breast carcinoma cell line MCF-7.</p><p><b>METHODS</b>MCF-7 cells were randomly divided into four groups depending on the culture solution used, the control group, cultured with 1640 medium not contained ZLJ; and the three ZJL groups cultured with medium contained low (1.5 mg/mL), moderate (3 mg/mL) and high (6 mg/mL) dosage of ZLJ crude drug respectively. The changes of cell proliferation were assessed by cell growth curve assay and methyl thiazolyl tetrazolium (MTT) assay. And the cell apoptosis was analyzed by flow cytometry, Hoechst 33342 staining and DNA ladder assay.</p><p><b>RESULTS</b>Compared with that in the normal control, the counts of cells in the three ZLJ groups were decreased significantly (P<0.05) at such time point as 24, 48, 72, 96, 120, and 144 h. Furthermore, apart from the comparison of the growth inhibition rate between the low and moderate dosage group at 24 and 72 h which were found to be no significant difference (P>0.05), the comparison f that among the three ZLJ groups appeared to be significant difference (P<0.05). The inhibitory effect of ZLJ on cell proliferation of MCF-7 was time- and dose-dependent; it could retard cells in G0/G1 cell phase; apoptosis of MCF-7 cell was induced by moderate and high dosage of ZLJ with revealing of apoptotic body and DNA ladder formation.</p><p><b>CONCLUSION</b>ZLJ shows cell proliferation inhibitory and apoptosis inducing effects on human breast cancer cell line MCF-7, and thus to realize its anti-tumor action.</p>