Radiotherapy-induced Increase In The 12-LOX And CCL5 Levels Of Esophageal Cancer Cells Results In THP-1-derived Macrophages That Promote Cancer Metastasis

Research purpose:A dioxygenase named 12-lipoxygenase(12-LOX)plays an important role in tumorigenesis and promotes angiogenesis and proliferation in several tumors,such as prostate tumor,breast tumors and melanoma.Radiotherapy could enhance the expression of 12-LOX in esophageal squamous cell carcinoma(ESCC).Two types of macrophage can be found in tumor microenvironment.One subtype is called M2,which accelerates tumor progression.However,the relationship between 12-LOX and macrophages is not well established.Here,we explored this interaction and its effect on ESCC to induce tumor progression.Methods and results:RT-qPCR and Western blot analyses were used to evaluate the mRNA and protein expression of 12-LOX and chemokine(C-C motif)ligand 5(CCL5)in ESCC after radiotherapy.CCL5 expression was increased by 12-LOX upregulation but suppressed by baicalein,a well-established inhibitor of 12-LOX.Further Western blot results showed that 12-LOX regulated the expression of CCL5 through the Akt/NFκB pathway.Our results also confirmed that CCL5 attracted and repolarized human myeloid leukemia mononuclear cells(THP-1)derived macrophages.We cultured THP-1-derived macrophages in the conditioned medium(CM)of Eca109 which is before and after radiotherapy,and found the re-polarization of macrophages to the M2 subtype in Eca109 CM after radiotherapy.The same results were found in Kyse150 cells.At the same time,macrophages were cultured in CM with Kyse150 cells upregulated 12-LOX or Eca109 cell line inhibited by baicalein.Compared with CM cultured macrophages cultured with Kyse150 or Eca109 in the control group,the results of RT-qPCR showed a trend of macrophage polarization to M2 or M1,respectively.In further experiments,ESCC co-culture with THP-1 derived macrophage led to strong cancer migration capacity.Finally,we used immunohistochemistry to assay the expression of 12-LOX,CCL5,CD68 in tissue sections of 40 patients with different stages of esophageal cancer from Qilu hospital(Shandong University,Jinan).Conclusion:The radiation-induced 12-LOX overexpression in ESCC upregulates CCL5 expression,thereby attracting THP-1-derived macrophages and promoting their polarization to M2 that enhances cellular metastasis.In addition,immunohistochemical results showed that high expression of 12-LOX,CCL5 and CD68 was associated with poor survival outcomes,and further multivariate analysis showed that 12-LOX was an independent prognostic factor for overall survival(OS).Significance:the significance of this study is reflected in two aspects.First,in theory,we discussed the relationship between 12-LOX and CCL5,and 12-LOX and macrophages.To the best of our knowledge,this is the first study to do so.Second,in terms of clinical application,we preliminarily discussed the correlation between 12-LOX and the prognosis of patients with esophageal cancer,and proved that 12-LOX is an independent prognostic factor,which laid a foundation for further clinical studies.