LIF/Stat3 Signaling Status During Stepwise Gastrocarcinogenesis And Its Potential Implications

Objective The aims of current study are to investigate the expression status of LIF(Leukemia inhibitor factor), Stat3(Signal transducer and activator of the transcription 3), VEGF(vascular endothelial growth factor)and Survivin in gastric cancer and to analyze the possible relationship between LIF abnormal expression and constitutive Stat3 activation of signaling pathway;blocking the expression of p-Stat3 in BGC 823 by JAK inhibitor AG490, to analyze the expression statuses of LIF, p-Stat3,VEGF and Survivin, so to explore the potential molecular mechanisms of gastric carcinogenesis and development.Materials and Methods Surgical specimens were selected from the gastric tissue bank of Liaoning Key Lab of Cancer genomics, Dalian Medical University. Human gastric cancer cell lines AGS and HGC-27 were ordered from American Type Culture Collection (ATCC, USA), MGC803 and BGC823 were kindly provided by Beijing Institute for Cancer Research, Peking University. The different gastric tissues were classified histologically as paramalignant and gastric cancer tissues and indicated by the methods of paraffin tissue array-based immunohistochemical staining. Paralleled Western-blotting and RT-PCR analyses were conducted to check the expression statuses of LIF, Stat3, VEGF and Survivin in four human gastric cancer cell lines. And after adding AG490 (80μmol/L) in BGC823 during cell culture, we checked the expression statuses of LIF, Stat3, VEGF and Survivin before and after adding the inhibitor by ICC, Western-blotting and RT-PCR. Statistical significance was determined by t test, X2 test and Spearman correlation test in this work and p<0.01 was considered statistically significant.Results1. The statuses of LIF, VEGF and Survivin expressions were increased distinctively in gastric cancer in comparison with that in paramalignant group. 2. Among 65 gastric tissue spots, 45(45/65, 69.23%) showed positive nuclear translocation of Stat3 and 43(43/65, 66.15%) were positive for LIF staining, which were essentially observed in gastric cancer tissues. Importantly, of the 45 nuclear Stat3 positive cases, 40 (40/45, 88.89%) were also positive for LIF staining, and there was a distinct correlation between nuclear positive staining of Stat3 and LIF(X2=33.762; p<0.01).3. VEGF overexpression and nuclear translocation of Stat3 appeared in great proportion of gastric cancer tissues (r=0.502; p<0.01) and similar phenomenon could also be observed in the examination of Survivin expression (r=0.533; p<0.01). Consequently, a significant correlation can be found between nuclear translocation of Stat3 and VEGF or Survivin expression.4. The level of mRNA was determined using RT-PCR analysis and we could detect different mRNA amount of LIF. LIF mRNA expression level was apparently higher in AGS than that in other three cell lines, while the levels of Stat3, VEGF and Survivin appeared similar among those four cell lines. Meanwhile, production of corresponding proteins were determined via Western-blotting analysis.The amounts of LIF, Stat3, VEGF and Survivin proteins remain similar among the four cell lines.5. Comparing the normal cell, p-Stat3, VEGF and Survivin downexpressions were evident in the cell with inhabitor AG490.While there was no change in expression of LIF.So p-Stat3 can up-regulate VEGF and Survivin.6. JAK inhibitor AG490 could evidently block LIF/Stat3 signaling pathway, and p-Stat3, VEGF and Survivin down-expressed, which probably show the importance of Stat3.The results told us we can use JAK inhibitor AG490 to facilitate apoptosis in gastric cancer therapeutics.Conclusion Our current study demonstrates constitutive Stat3 activation and frequent expressions of its downstream genes, VEGF and Survivin in gastric tumors and cell lines. The above phenomena were closely associated with LIF expression. In accompany with LIF up-regulation, cytoplasmic Stat3 is translocalized into the nucleus, which is correlated with VEGF and Survivin expression. Our results reveal a positive relationship between LIF expression and Stat3 activation as well as up-regulated VEGF and Survivin expression in gastric cancers. Consequently, gastric cancer cell are conferred on strong ability of angiogenesis and apoptosis suppression.