Effects And Related Mechnisms Of S100A4on HBL-100and MCF-7Cell Lines

Backgrounds and objectivesIn the world it is about10%of women suffer from breast cancer, abouthalf of patients will die from it, so this disease has caused a great threat to thehealth of women.S100A4is a member of S100cadherin superfamily, it consists101amino acid residues,its molecular weight is about11.5KD, it can exist innuclear,cytoplasmic and the cellular microenvironment. its overexpressioncan promote the development of rheumatoid arthritis, kidney fibrosis,liverfibrosis and other diseases, it is also close related to tumor development.Wnt signaling pathway is a relatively conservative pathway in theprocess of biological evolution,it was involved in the regulation of cellproliferation,differentiation,apoptosis and other biological processes. Itcantains the classic signaling pathway which depend on β-catenin and thenon-classical signaling pathway which doesn’t rely on β-catenin. β-cateninis a key molecule in the classical pathway.Its intracellular level can indicatethe activity of the Wnt pathway,it was degradated by GSK-3β, APC andAxin complex; wnt/c-Jun NH2-terminal terminal kinase (JNK) pathway isan important part of non-canonical Wnt pathway.The present study showed that the Wnt signaling pathway is intimately linked with tumor occurrenceand development.There are abnormal activation of the Wnt pathway inbreast cancer.Methods1. Amplification of the recombinant adenovirus AdS100A4anddetermine the optimal virus titer.2.Through RT-PCR and Western Blot to identify the expression ofendogenous S100A4in HBL-100and MCF-7cells lines and to detect thechange of it after AdS100A4infection.3. After infection of HBL-100and MCF-7with appropriate amount ofvirus respectively, to detect cell proliferation by MTT, to assay cellmigration by wound healing assay and Transwell migration, to detect cellinvasion by transwell invasion experiment and to detect cell apoptosis byHoechst staining.4. Through RT-PCR and/or Western blot to detect the levels ofintracellular β-catenin,GSK3β phosphorylation and c-Myc after cell linesinfected with AdS100A4, to explore whether the classic Wnt signal pathwayis involved in the role of S100A4on breast cancer.5. Through Western blot or PCR to detect the level of JNKphosphorylation and c-Fos mRNA levels in these cell lines,to explorewhether the Wnt/JNK pathway is involved in the role of S100A4on breast cancer.Results1.There were expression of green fluorescent protein in HEK293cellinfected by AdS100A4, the infection rate at72h was more than90percent;the AdS100A4titer was2×1011IU/ml.2. S100A4mRNA and protein were not found in HBL-100; but theywere found in MCF-7.3. The expression of S100A4in these two cells are significantlyincreased after infection with AdS100A4. The prompt that S100A4genecarried by the AdS100A4can express successfully in the target cells,indicating that the experimental interventions is effective.4. MTT assay showed that:1) The proliferation of cell line HBL-100and MCF-7increased by52.8%(P <0.05) and50.5%respectively than theircontrol group after infection of AdS100A4four days (P <0.05), suggestingthat S100A4can promote proliferation of HBL-100and MCF-7.Suggestingthat S100A4can promote the proliferation of HBL-100and MCF-7celllines.5.Wound healing and Transwell migration assay showed that:1) Thewound healing rate of HBL-100and MCF-7treated by AdS100A4in24hour increased by73.6%(P <0.05) and64.8%(P <0.05), respectively, thanthe control group;2) The trans-membrane celles was about1.73times (P <0.05) and1.95times(P <0.05)than the control group; These resultssuggested that S100A4can promote the cell migration of these two celllines.6. Transwell invasion experiment showed that:1) The number ofpenetrating HBL-100cells treated by AdS100A4is about5which was nodifference with the control group number of5;2) The number of penetratingMCF-7cells treated by AdS100A4was approximately2times of the controlgroup,(P <0.05). These results suggest that S100A4had no significant effecton the invasion of HBL-100, but it can significantly enhance the invasionability of MCF-7.7.Hoechst stain showed that:1) The24h apoptosis ratio of HBL-100and MCF-7treated by AdS100A4were5.5%and3.62%respectively, therewere no significant difference with the GFP group (P>0.05);2) The48hapoptosis ratio of HBL-100and MCF-7treated by AdS100A4decreasedby29.9%(P <0.05) and35.8%(P <0.05) than the control group. Theseresults prompt that S100A4can inhibit apoptosis of these cells.8.The effect of S100A4on Wnt/β-catenin:8-1.Western Blot showed that:1) The β-catenin calibration gray valuesin HBL-100and MCF-7treated by AdS100A4were increased by49%(P<0.05) and55%(P <0.05) than their control group respectively; The c-Myccalibration gray values in HBL-100and MCF-7treated by AdS100A4were increased by38%（P＜0.05）and30%（P＜0.05）than their controlgroup,respectively.This suggested that S100A4can active the Wnt/β-catenin signal pathway.8-2.RT-PCR shows that:S100A4had no significant effect on thetranscription of β-catenin in HBL-100and MCF-7cell lines; Western blotshow that the P-GSK3β calibration gray of HBL-100and MCF-7infected byAdS100A4increased by49%(P <0.05) and55%(P <0.05)。It can increasethe intracellular level of β-catenin:1)it unrelated to its transcriptional;2)One mechanism is through the increase of GSK3β phosphorylation, thusinhibiting the degradation of it.8-3.Analyzes show that:1) The β-catenin calibration gray of HBL-100treated by AdS100A4was36.6%lower than that of MCF-7(P <0.05);2) TheP-GSK3β calibration gray of HBL-100infected by AdS100A4was35.7%more than that of MCF-7(P <0.05);3) The c-Myc calibration gray ofHBL-100infected by AdS100A4was17.5%less than that of MCF-7(P<0.05). This suggests that S100A4activate the role of Wnt/β-cateninpathway in MCF-7more than HBL-100.All of this suggests that S100A4can activate the classical Wntsignaling pathway in HBL-100and MCF-7, this role is stronger in MCF-7.Itactivated this pathway is through increasing the level of P-GSK3β.9. The effect of S100A4on the Wnt/JNK signal pathway 9-1.Western Blot and PCR results showed that S100A4had no effecton the P-JNK and c-Fos mRNA levels in HBL-100;9-2.Western Blot and PCR showed that the P-JNK and c-Foscalibration gray of MCF-7treated by AdS100A4increased39%(P <0.05)and32.3%(P <0.05) respectively.This suggested that S100A4had no effect on the activity of the Wnt/JNK signaling pathway in HBL-100, but it can activate the Wnt/JNKsignaling pathway in MCF-7cell line.Conclusions1. AdS100A4can be successfully amplified in HEK293cell.2. HBL-100has no S100A4expression or its expression level isbelow the method detection limit; there is S100A4expression in MCF-7.3. AdS100A4which carryes S100A4gene was successfully expressionand upreglate S100A4protein level in its target cells (HBL-100and MCF-7cell lines).4. S100A4could significantly promote the proliferation, migration ofHBL-100cell and inhibit its apoptosis, but have no effect on its invasionability.5. S100A4can also significantly promote the proliferation andmigration of MCF-7and inhibit its apoptosis, it also can promote theinvasion ability of MCF-7.This suggested that S100A4may be involved in breast cancer progress.6. S100A4can activate the Wnt/β-catenin signal pathway in HBL-100and MCF-7cell lines,which is one of the mechanisms of S100A4effects onthese cell lines;it also can increase the level of β-catenin in these cellesthrough increased the phosphorylation of GSK3β （inhibition thedegradation of intracellular β-catenin）,nothing to do with the transcriptionof β-catenin.7. The role of S100A4effect on HBL-100cell does not involve the Wnt/JNK pathway.8. S100A4can active Wnt/JNK/c-Fos pathway in MCF-7cell,which may mediate the role of S100A4on MCF-7cell.