Anti-proliferation effects of dammar-24-ene-3β-acetate-20S-ol on gastric cancer cells / 医学研究生学报

Objective Based on the previous research that the ethanolic extract from traditional Chinese medicine fructus forsythiae (Lianqiao) can obviously inhibit cancer cells in vitro, the article aimed to investigate the anti-proliferation effects of dammar-24-ene-3β-acetate-20S-ol (DM) extracted from fructus forsythiae on gastric cancer cells and its mechanism.Methods MTT assay was used to assess the anti-proliferation effects of DM on gastric cancer cells including SGC-7901, BGC-823, and MKN-45 in vitro.There were MKN-45 control group and its low dose and high dose groups, BGC-823 control group and its low dose and high dose groups, SGC-7901 control group and its low dose and high dose groups in the experiment.Flow cytometry was used to analyze the cell apoptosis rate.Cellquest software was used to analyze the results and record the ratio of cells at different cycles.DCFH-DA probe was applied to detect the ROS levels of blank control group, docetaxol group and DM group.The reaction system of microtubule assembly test was set with 10?mol/L docetaxol, 50 or 100 μmol/L DM final density and no medicine in blank control group.The readings of UV spectrophotometer were recorded.Microtubule assembly assay and microtubule immunofluorescence staining were applied to investigate the effects of DM on microtubule system.Results The inhibition ratio of 50 μg/L DM on the proliferation three gastric cell lines were all above 80%, with IC50s of MKN-45 11.72±1.35 μg/mL, BGC-823 17.19±0.82 μg/mL, SGC-7901 7.55±0.79 μg/mL.8 days′ low density culturing at 48 hours after 2 μg/mL DM treatment, compared with control group, the number of cell clones significantly reduced without much change in clone size, while 48 hours after 10 μg/mL DM treatment, besides a few clones of BGC-823, there were just several megascopic clones of SGC-7901 and MKN-45.In comparison with apoptotic cell ratio in MKN-45 control group[(21.1±2.5)%], its low dose group and high dose group resulted in significant rise of apoptotic cell ratio[(25.1±1.3)% and (55.2±2.3)%] (P0.05).In comparison with MKN-45 control group, the ratio of cells at S phase decreased in its low dose group[(14.5±2.7)% vs (12.3±3.3)%,P>0.05].In comparison with BGC-823 control group, the ratio of cells at S phase increased in its low dose group[(12.2±5.4)% vs (20.2±2.1)%,P<0.05].In comparison with SGC-7901 control group, the ratio of cells at S phase increased in its low dose group[(21.5±3.8)% vs (31.3±2.6)%,P<0.05].From the detection of intracellular active oxygen after DM treatment, dose-dependent ROS level increased in all three cell lines 48 hours after 10μg/mL and 50μg/mL DM treatment.From the results of microtubule immunofluorescence staining, 48 hours after the treatment of IC50 docetaxol and 10μg/mL DM, the fluorescence signals were in local concentration and disorder.Conclusion Dammar-24-ene-3β-acetate-20S-ol demonstrated anti-proliferation effects due to the apoptosis induced by cell cycle arrest at S phase.

Inhibition effects of fungal immunomodulatory proteins from Ganoderma spp .on different gastric cancer cell lines in vit ro / 药学实践杂志

Objective To study the cell proliferative effects of fungal immunomodulatory proteins from Ganoderma spp . on 26 gastric cancer cell lines in vitro .Method 26 human gastric cancer cell lines were treated with FIPs by MTS assay .The average optical density (OD) in 490 nm and inhibition rate (GI50 )was counted by Universal Microplate Spectrophotometer . Results Three FIPs showed similar profiling in 26 human gastric cancer cell lines after 72 h treatment in cell proliferation as-say ,which except for NUGC-4 and OCUM-1 did not showed obvious anti-proliferative effect ,the other 24 human gastric cell lines showed some anti-proliferative effects ,especially for 7 cell lines(NUGC-3 ,GTL-16 ,HGC-27 ,IM95m ,SNU-638 ,SNU-216 and SNU-5) showing strong potency ,with their GI50 less than 50 μg/ml .Conclusion FIPs showed strong anti-prolifera-tive effects in some human gastric cancer cell lines in vitro ,which had potential to be further developed as anti-gastric cancer drugs .

Effect and mechanism of rmhTNF on gastric cancer cell lines with differentΔ133p53 status / 中国癌症杂志

Background and purpose: Little about the function of p53 isoforms in gastric cancer was reported. This study was designed to explore the role ofΔ133p53 in the effect of recombinant mutant human tumor necrosis factor (rmhTNF) on gastric cancer cells, and provide a new basis for the diagnostics and therapeutics of gastric carcinoma. Methods: MKN45 (withΔ133p53 expression) or SGC7901 (withoutΔ133p53 expression) cells were treated with rmhTNF of different concentrations only or combined with 5-FU (a traditional gastric cancer cellular killer), and the growth inhibition rate and apoptosis was detected by CCK-8 and lfow cytometry. mRNA expressions ofΔ133p53, Gadd45αand CyclinB1 were measured by nested reverse transcription-polymerase chain reaction (nRT-PCR) or real-time polymerase chain reaction(RT-PCR). Results:On MKN-45 cells with positiveΔ133p53 expression, the inhibitory effect of rmhTNF was signiifcant, the inhibition rates of 50 and 500 IU/mL rmhTNF were 24.82%, 72.33%after culturing for 24 h (t=-9.558, P0.05). In apoptosis test, the apopto-sis-enhancing effect of rmhTNF was signiifcant on MKN45 cells, and the apoptosis-enhancing effect of 5-FU was fur-ther promoted signiifcantly by rmhTNF, the apoptosis of rmhTNF (50 IU/mL), rmhTNF (50 IU/mL) combined with 5-FU (25 μg/mL), rmhTNF (500 IU/mL) combined with 5-FU (25 μg/mL) were 18.20%, 48.66%, 59.83%, separately, after culturing for 24 h (F=123.931, P<0.05). In mRNA measurement, down-regulation ofΔ133p53 and CyclinB1, up-regula-tion of Gadd45αwere signiifcant in MKN45 cells treated by rmhTNF alone or combined with 5-FU. In nRT-PCR anal-ysis, the mRNA levels ofΔ133p53 were relatively 0.886, 0.499, 0.330, 0.161 (F=240.927, P<0.01);In real-time PCR analysis, the mRNA levels of Gadd45αwere 1.227, 1.694, 3.394, and the mRNA levels of CyclinB1 were 1.221, 0.722, 0.316, relatively. The expression ofΔ133p53 was positively related to CyclinB1 (r=0.977, P<0.01), but negatively re-lated to Gadd45α(r=-0.950, P<0.01). Conclusion:InΔ133p53 positively expressed MKN45 cells, rmhTNF showed as an effective tumor inhibitor and an enhancer of 5-FU as well, and this effect might be helped by two p53 down-stream molecules CyclinB1 and Gadd45α. The results suggest thatΔ133p53 might be a key target for the biological effect of rmhTNF against gastric cancer.

Sequence Analyses of Aberrant FHIT Transcripts in Gastric Cancer Cell Lines / 대한소화기학회지

BACKGROUND/AIMS: The fragile histidine triad (FHIT) gene located at chromosome 3p14.2, is a candidate tumor suppressor gene often involved in various tumors. Homozygous deletions, lack or reduced expression of FHIT protein, and alteration of its transcription were frequently observed in several types of primary human cancers and cell lines. In the present study, we examined the expression profiles of aberrant FHIT transcripts to explore the role of FHIT gene in gastric carcinogenesis. METHODS: In 6 gastric cancer cell lines, nested reverse transcription-polymerase chain reaction (RT-PCR) and cDNA sequence analyses were performed to detect and characterize the aberrant FHIT transcripts. RESULTS: In addition to the wild-type FHIT transcript, small-sized transcripts with various numbers and lengths were observed in all of the cell lines examined. cDNA sequence analysis confirmed that different types of truncated transcripts included exonic deletions, insertions of intron 5 sequences between exons, and combinations of both. Most of these transcripts lacked exon 5 in which translation initiation codon is located. Aberrant transcripts with partial exonic deletions due to activation of cryptic splice sites were also observed in 5 cell lines. Additionally, multi-step splice patterns indicative of additional downstream processing, were observed in several cancer lines. CONCLUSIONS: These results suggest that the aberrant FHIT transcripts in gastric cancer cell lines results from faulty splicing, including exon skipping, selection of cryptic splice site, and additional downstream splice processing.

CpG methylation as a basis of specific loss of normal epithelial cell-specific-1 gene in gastric tumor / 中华消化杂志

Objective To investigate the expression of normal epithelial cell-specific-1(NES1) gene in normal gastric epithelial cells and different gastric cancer cell lines and the effects of 5-aza-2-de- oxycytidine(5-aza-dC)on the expression of NES1 gene.Methods Expression of NES1 mRNA in five gastric cancer cell lines(MKN-28,SGC-7901,AGS,MKN-45 and HGC-27)and normal human gastric epithelial cells were detected by real-time PCR.After treatment with 5-aza-dC,a DNA methyltransferase inhibitor,the expression of NES1 mRNA in gastric cancer cell lines was detected by real-time PCR. DNA methylation status of NES1 gene was assayed by methylation-specific PCR(MSP).Results The expression of NES1 mRNA was decreased in all gastric cancer cell lines.A strong correlation between ex on 3 hypermethylation and loss of NES1 mRNA expression in gastric cancer cell lines was noted.5-aza dC treatment of NES1-nonexpressing tumor cell lines resulted in a dose-dependent induction(SGC-7901, MKN-45,MKN-28 and AGS)and increase (HGC-27) of NES1mRNA expression in gastric cancer cells. Conclusions The study suggested that hypermethylation was a responsible factor for tumor-specific loss of NES1 gene expression in gastric cancer cells.Treatment of gastric cancer cell lines with a demethylating agent led to reexpression of NES1 suggesting an important role of hypermethylation in loss of NES1 gene expression.

Chromosomal Alterations in Gastric Cancer Cell Lines Detected by Comparative Genomic Hybridization / 대한암학회지

PURPOSE: There are only a few cytogenetic studies in gastric cancer and so far no consistent specific chromosomal abnormalities have been described. In this study, we have used comparative genomic hybridization (CGH), a powerful molecular cytogenetic technique for detecting changes of the copy number throughout the genome, to screen for genetic alterations in gastric cancer cell lines. The CGH results were compared with those derived from G-banding and chromosome painting. MATERIALS AND METHODS: Conventional cytogenetic analysis was performed on five human gastric cancer cell lines, AGS, SNU-1, SNU-16, SNU-620, and SNU-719, by a G-banding staining technique. In CGH, equal amounts of differently labeled DNA from the cell lines and normal reference DNA were hybridized simultaneously to normal metaphase chromosomes. They were visualized by different fluorochromes, and the signal intensities were quantitated separately as gray levels along the single chromosomes. The over- and under- represented DNA segments were determined by computation of ratio images and average ratio profiles. To confirm the CGH results, fluorescence in situ hybridization (FISH) with chromosome specific painting was performed using indirectly labeled chromosome specific paints. RESULTS: Complex unbalanced chromosomal aberrations that could not be identified reliably by conventional cytogenetics in gastric cancer cell lines were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome specific probes. In gastric cancer cell lines, gains of DNA copy number were more common than losses. Gains were detected on 1p, 1q, 2p, 3q, 6p, 7q, 10q, 11p, and 19q, and losses were observed on 4p, 4q, 5q, 12p, 12q, and 18q. Interestingly, all the five gastric cancer cell lines tested showed gain of DNA copy number on the chromosome 20, suggesting an existence of oncogene. CONCLUSION: Conventional cytogenetics, CGH, and FISH using painting probes represent complementary approaches that, when employed in combination, could greatly facilitate the comprehensive analysis of chromosomal imbalances in gastric cancer cell lines. Our results suggest the existence of an oncogene or oncogenes on chromosome 20 that play a role in the development and/or the progression of gastric carcinogenesis.

Antitumor mechanism of ursolic acid on human gastric cancer cell lines SGC7901 in vitro / 中草药

Objective To investigate the potential mechanism of inhibition of ursolic acid(UA) on growth of human gastric cancer cell lines SGC7901 in vitro.Methods SGC7901 cells were cultured in vitro,MTT assay was used to observe the effect of UA on growth of SGC7901 cells in various concentrations for different times.After SGC7901 cells were treated by 0—40 ?mol/L UA for 24 h,morphological changes were observed by inverted microscope.Apoptotic changes were detected by fluorescence microscopy and flow cytometry (FCM).Protein expressions of Bcl-2 and Bax were determined by Western blotting.Results UA(20—40 ?mol/L) could significantly inhibit the growth of SGC7901 cells in a(dose-and) time-dependent manner,the IC_(50) value of UA for SGC7901 cells for 12,24,36,and 48 h were((57.50?)1.18),(34.28?2.05),(27.54?1.11),and(24.83?1.02) ?mol/L,respectively.After UA((20—)40 ?mol/L) treatment for 24 h,SGC7901 cells turned round and floated at different levels;SGC7901 cells were arrested at G_0/G_1 phase and apoptosis was induced,and the apoptotic rate was increased along with the increase of UA concentration.Meanwhile Bcl-2 protein expression decreased,whereas Bax protein expression unchanged.Conclusion UA has a stronger antitumor effect on SGC7901 cells.Cytotoxic effect,proliferation inhibition,and apoptosis may be involoved in the mechanism of UA,and the apoptosis caused by UA may be enhanced by decrease of Bcl-2 protein expression.