| Objective To investigate the bridge role of IL-8on leptin induced EMT inbreast cancer MCF-7and SK-BR-3cells in vitro.To explore the relativitybetween leptin and IL-8accelerating progression of breast cancer in vivoand provide experimental data for therapeutic strategies of breast cancer.Methods1. Western blot and Immunofluorescence assay were applied toidentify the expression of leptin receptors OB-Rb and OB-Rt in humanbreast MCF-7, SK-BR-3and MD-MB-231cells. Leptin-inducedmorphological changes of MCF-7, SK-BR-3and MD-MB-231cells weredetermined by microscope. The effect of leptin on expression ofEMT-related markers E-cadherin,vimentin and fibronectin were detected byWestern blot.2. The influence of leptin on IL-8mRNA or protein expression ofMCF-7and SK-BR-3cells was detected by QRT-PCR and western blot.3. Specific blocking antibodies were added to neutralize IL-8 secrected from MCF-7and SK-BR-3cells. Western blot andImmunofluorescence assay were hired to investigate the effect of leptin onexpression of E-cadherin, vimentin and fibronectin. Cell scratch andtranswell assay were executed to check the effect of leptin on migration andinvasion potential of MCF-7and SK-BR-3cells.4. Western blot was applied to determine the effect of leptin-leptinObR axis on activation of JAK/STAT3, MAPK/ERK1/2and PI3K/AKTsignaling pathways and secretion of IL-8in MCF-7and SK-BR-3cells.Pharmacological inhibitors were employed to block JAK/STAT3andPI3K/Akt signaling pathways. QRT-PCR and western blot were applied toidentify the influence of leptin on IL-8, E-cadherin, vimentin andfibronectin expression in MCF-7and SK-BR-3cells.5. Immunohistochemical analysis were constructed to detect theexpression of ObR, Leptin, IL-8, E-cadherin and vimentin in benign breasttissues, invasive breast carcinoma without metastasis and carcinoma withits lymph node metastases.6. Nude mice breast cancer xenograft model was established toresearch effects of leptin in vivo. MCF-7cells were inoculated into themammary fat pads of female nude mice (5to7-week-old). When the tumordiameter reached0.5mm on day15, mice were injected with PBS or leptinuntil day30respectively. Diameter of tumor were measured per5days.When partial mice were put to death, tumor, lung and liver tissues were surgically removed, tumor volumes and weights were determined.Immunohistochemical analysis were subjected to examine IL-8, E-cadherin,Vimentin and Ki67expression of tumor sections. Lung and liver metastasiswere measured by HE staining of lung and liver tissues. Survival time ofmice were also monitored.Results1. Western blot and Immunofluorescence assay showed that bothOb-Rb and Ob-Rt were presented in MCF-7, SK-BR-3and MD-MB-231cells. EMT-related morphological changes of leptin-treated MCF-7andSK-BR-3cells emerged included cell-cell junction dissolution, loss ofapical-basolateral cell polarity, increased formation of pseudopodia.Morphologies of leptin-treated MDA-MB-231cells did not changecompared with untreated cells. Besides, Western blot showed MCF-7andSK-BR-3cells exhibited a significant upregulation of mesenchymalmarkers such as vimentin and fibronectin, accompanied by apparentdecrease of epithelial marker such as E-cadherin, which indicated a switchfrom an epthelial to a mesenchymal-like phenotype. MD-MB-231cells hadno corresponding changes of EMT when treated with leptin. MCF-7andSK-BR-3cancer cells were chosen to subsequent reasearch.2. The results of QRT-PCR and Western blot showed that leptininduced IL-8secretion of MCF-7and SK-BR-3in a dose andtime-dependent manner.3. To further estimate the direct role of IL-8in the process of leptin-induced EMT in breast cancer, IL-8secreted from MCF-7andSK-BR-3cells were neutralized with IL-8blocking antibodies. Westernblot and Immunofluorescence assay showed significant upregulation ofE-cadherin, accompanied by reduction of vimentin of MCF-7and SK-BR-3cells in the group united application of leptin with IL-8specific antiboby.Cell scratch and transwell assay showed that treatment with IL-8specificantiboby significantly inhibited the migration and invasiveness induced byleptin in MCF-7and SK-BR-3cells.4. By Western blot we found that leptin activated classicalJAK/STAT3, MAPK/ERK1/2, PI3K/AKT signaling pathways. When specialantibobies against ObR was added to MCF-7or SK-BR-3cells ahead ofleptin treatment, IL-8secreted from MCF-7and SK-BR-3cells induced byleptin were down-regulated, the activity of leptin-induced JAK/Stat3andPI3K/Akt signaling pathways were completely abolished owing todestruction of leptin-OBR axis, whereas the presence of specific antibobiesdid not change sustained activation of MAPK/ERK1/2signaling pathway.QRT-PCR and Western blot were hired to investigate whether leptininduced secretion of IL-8from MCF-7and SK-BR-3cells via JAK/STAT3and PI3K/AKT signaling pathways. The results showed that leptin-inducedmRNA and protein of IL-8were abolished when PI3k/AKT signalingpathway was suppressed, whereras blockage of JAK/STAT3could notrestore the protein changes of IL-8achieved by leptin treatment, Moreover, the effects of signaling pathway inhibitors on leptin-inducedprotein of Vimentin, Fibronectin and E-cadherin, biochemical hallmarks ofEMT were similar to that for IL-8protein.5. Immunohistochemical analysis was employed to detect expressionof leptin, ObR, IL-8, E-cadherin, vimentin in pathological specimens ofbreast hyperplasia tissues. The results showed that the staining intensity ofObR, Leptin, IL-8,vimentin in invasive breast carcinoma without axillarylymph node metastases was lower than in LNM and stronger compared tothat in benign breast tissue, accompanied with contrary result ofE-cadherin.6. The results of nude mice xenograft model showed that tumorvolume of mice treated with leptin was significantly larger than PBSinjection. When partial mice were put to death, tumor was surgicallyremoved, it was found that treatment with leptin led to a distinct increase intumor volume and weight compared with group PBS. The rusults of HEstaining showed that leptin injection led to more massive metastasis in thelungs and livers of tumor-bearing mice as compared with PBS injection.The results of in situ immunohistocheminstry showed that the expression ofIL-8, Ki-67, vimentin was enhanced in leptin treated group, and E-cadherinwas strikingly reduced. We discovered that leptin reduced the survival ofmice as well.Conclusion1. Leptin could promote EMT of breast cancer MCF-7and SK-BR-3cells, which might associated with secretion of IL-8viaactivation of PI3K/Akt signaling pathway.2.The expression of ObR, IL-8, EMT-related marker in invasive breastcarcinoma with its lymph node metastases were clearly higher than breastcarcinoma without lymph node metastasis and benign breast tissue.3. In tumor-bearing mice, leptin could increase the growth of tumor,promote metastasis in the lungs and livers, reduce the survival of mice andenhance IL-8expression of tumor tissues. |
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