The Study Of PGE2 Secreted By Cervical Cancer HeLa Inhibits CCL5 Expression Of The Macrophage RAW

Objective: Cervical cancer is one of common gynecologic malignant tumor in the world, behind breast cancer, moreover its attack rate both goes up year by year and makes more youthful. Due to the existence of immune inhibitory factor in tumor microenvironment, for example, prostaglandin E2(prostaglandin E2, PGE2), makes many inflammatory cytokines can’t work normally, but play a role of promoting tumor effect. Study shows that inflammation is closely related to the development of tumor, in order to further study the relationship between inflammation and tumors in cervical cancer victims, He La cells supernatant was collected as tumor condition medium(TCM), to evaluate whether CCL5 secretion by macrophages RAW was inhibited, and whether the inhibition effect is induced by the PGE2 of TCM. Subsequently, we explore the mechanism that CCL5 expression of RAW cells was inhibited by PGE2, all these results will provide new insights for the clinical treatment of cervical cancer.Methods:1 Preparation of tumor conditioned medium(TCM) : 0.6 x 106 /ml of He La cells were cultured with complete medium for 72 h, and then the supernatant was collected, and filtered by 0.45μm filter, which was kept in-20 for standby, namely for TCM.℃2 Macrophages RAW were stimulated with different doses of(0, 200, 400, 600μl) TCM and lipopolysaccharide(LPS)(1μg/ml), and then protein and m RNA expressions of CCL5 in RAW cells were detected by ELISA and q RT-PCR, respectively.3 Protein and m RNA expressions of CCL5 in RAW cells were evaluated by ELISA and q RT-PCR respectively, following stimulation by different doses(0, 10-13, 10-12, 10-11, 10-10 M) of P GE2 and LPS(1μg/ml).4 Protein and m RNA expressions of CCL5 by RAW cells were detected by ELISA and q RT-PCR respectively, after PGE2 synthesis was blocked by inhibitor NS-398.5 Protein and m RNA expressions of CCL5 by RAW cells were detected by ELISA and q RT-PCR respectively, after PGE2 signaling was blocked by various PGE2 receptor antagonists including: 10μM SC51322(EP1 antagonist), 3μM AH6809(EP2 antagonists), 30μM AH23848(EP4 antagonists) and AH6809+AH23848, respectively.Results:1 After macrophages RAW was stimulated by different doses of TCM and LPS, protein and m RNA expressions of CCL5 by macrophages RAW were decreased with a dose-dependent manner. Compared with LPS group, significant differences were found in each groups(P<0.05).2 Protein and m RNA expressions of CCL5 by macrophages RAW were declined following different doses of PGE2 and LPS(1μg/ml) stimulation. Different doses of PGE2 group compared with LPS group, significant differences were found in each groups(P<0.05).3 The inhibition of CCL5 expressions induced by TCM was reversed after PGE2 synthesis in TCM was blocked by inhibitor NS-398, suggesting that PGE2 is an important component in TCM, and negatively regulated on CCL5 expression.4 We blocked these PGE2 receptors individually with SC51322(a selective EP1 antagonist), AH6809(the EP2 receptor antagonist) and AH23848(a selective antagonist for EP4) prior to LPS and TCM stimulation. EP1 antagonist had no effect on TCM-mediated CCL5 inhibition whereas EP2 and EP4 antagonists each could partially block the inhibitory effect of TCM on CCL5 protein and m RNA expression. More importantly, blocking both EP2 and EP4 receptors with AH23848 and AH6809 could completely abolish the inhibitory effect of TCM on CCL5 protein and m RNA expression, further demonstrating that the PGE2 secreted from cervical cancer He La cells mediates the inhibition through binding EP2 and EP4 receptors. Significant differences were found in Med(medium) and LPS, LPS and LPS+TCM, LPS+TCM and LPS+TCM+AH6809, LPS+TCM+AH23848, LPS+TCM+AH 6809+AH23848, respectively(P<0. 05). Conclusion: Tumor conditioned medium TCM which was derived from cervical cancer He La cells, TCM inhibits CCL5 protein and m RNA expression. According to the results, PGE2 of TCM is responsible for the inhibition of CCL5 expression. Thus, we use different doses of PEG2 to stimulate macroph-ages RAW and detect CCL5 expression. In order to further define the inhibit-ion effect of PGE2, we used PGE2 inhibitors NS-398 to block the generation of PGE2 and PGE2 receptor antagonist to block PEG2 signal pathway, which did reverse inhibitory effect of CCL5 expression. All those results remindered that the PGE2 secreted by cervical cancer He La cells inhibit protein and m RNA expression of CCL5.