| Objective: To explore the effect of IL-17 on apoptosis of myeloid-derived suppressor cells(MDSCs) and investigate the role of ERK1/2; to confirm whether IL-17 affect tumor growth by regulating apoptosis of MDSCs in Lewis lung carcinoma tumor-bearing mice.Method:(1) Lewis lung carcinoma(LLC) tumor bearing models were constructed with Lewis cells s.c. MDSCs were isolated from spleen in tumor-bearing mice and cultured in RPMI 1640 culture medium. Then MDSCs were treated with IL-17. Annexin V and PI was used to detect apoptosis of MDSCs. For further analyzing apoptosis of MDSCs, western-blot was also used to detect the expression of anti-apoptotic protein BCL-2 and phosphorylation of ERK1/2.(2) ERK1/2 inhibitor(U0126) was added in culture medium to pre-treat MDSCs. Then IL-17 was added later. Apoptosis of MDSCs was detected by Annexin V and PI and the expression of BCL-2 determined by western-blot.(3) Tumor-bearing mice were injected with IL-17 i.p to observe the growth of tumor. Weight and volumn of tumor were recorded and calculated. when the mice were sacrificed. The expression of Ki67 was detected by q RT –PCR.Detecting the percentage of DC, Macrophage, CD4+T and CD8+T cells by FCM. Immunofluorescence was used to confirm the counts of MDSCs in tumor. q RT –PCR was used to detect the relative expression of i NOS.(4) MDSCs were isolated from IL-17 treated tumor-bearing mice by MACS.Western-blot was used to detect the expression of p-ERK1/2 in MDSCs. MDSCs were maintained in RPMI 1640 culture medium. Annexin V and PI was used to detect the apoptosis of MDSCs. ARG-1 assay kit was used to detect the activity of ARG-1 of MDSCs. BCL-2 expression was determined by Westernblot.(5) Tumor-bearing mice treated with IL-17 were injected with gemcitabine(GEM) and then were transferred i.v. by MDSCs pre-treated with U0126.Tumor volumes and Tumor weights were detected when the mice were sacrificed. Detecting the expression of i NOs by q RT-PCR. Then MDSCs were isolated from tumor-bearing mice and cultured in 1640 culture medium. Apoptosis of MDSCs were detected by Annexin V and PI. ARG-1assay kit was applied for ARG-1 activity detection and Western- blot was used to detect BCL-2 expression level.Results:(1) MDSCs treated with IL-17 for 24 h, percentage of live cells of MDSCs were(45.33±1.556)% in control group and were(59.55±2.831)% after IL-17treatment(P<0.001). The later apoptosis of MDSCs were(22.87±1.349)% in control group and were(13.70±1.003)% after IL-17 treatment(P<0.01).Results of western blot showed that the expression level of p-ERK1/2(P<0.001) and anti-apoptotic protein BCL-2(P<0.01) significantly increased after IL-17 treatment.(2) Successively adding U0126 and IL-17 to culture system of MDSCs with or without U0126 treatment(7.5μM). Compared with percentage of live cells of MDSCs(39.08±2.718%) without U0126 treatment in 24 h, percentage of live cells of MDSCs were(15.57±1.009)% in U0126 treatment group(P<0.001).The later apoptosis of MDSCs were(8.007±1.777)% without U0126 treatment and were(26.95±1.179)% after U0126 treatment(P<0.001). Results of western blot indicated anti-apoptotic protein BCL-2 expressed in a lower level after U0126 treatment.(3) Compared with PBS treatment group, IL-17 treatment acceletated the growth of tumor. The weights of tumor in PBS group were 1.886±0.2020 g, while the weights in IL-17 treatment group were 3.480±0.4586 g. IL-17 treated tumor bearing mice also exhibited a significantly higher level of Ki67(P<0.05).The results of FCM suggested that the counts of MDSCs were(2.187±0.2541)×107 and the percentage were(13.40±0.3606)% of spleen(P<0.001)in PBS group; the counts of MDSCs were(3.967±0.1133)×107and the percentage were(24.43±1.1240)% of spleen(P<0.001)in IL-17 treatment group. IF indicated the counts of MDSCs increased in tumor tissues, the m RNA expression level of i NOS was also enhanced by q RT-PCR(P<0.05)of tumor tissues in IL-17 treatment group.(4) Compared with PBS treatment group, the results of western blot displayed a dreamtically upregulation in p-ERK1/2 and BCL-2 of MDSCs in IL-17 treatment group. Culturing MDSCs isolated from tumor bearing mice with IL-17 treatment for 24 h,the percentage of live cells were(44.23±3.477)%in PBS group and were( 59.33±2.718) % in IL-17 treatment group(P<0.05).The later apoptosis of MDSCs were(33.28±4.104)% in PBS group and were(19.26±2.494)% in IL-17 treatment group(P<0.05).(5) Compared U0126 group with control group, the counts of MDSCs decreased by 3.6%(P<0.05)in spleen and decreased by 5.3% in tumor tissues in U0126 group. Tumor volumes and tumor weights decreased in U0126 groups.Tumor weights were 2.948±0.7537 g in control group and were0.7830±0.1597 g in U0126 group. Ki67 expressive level also showed a reducing tendency( P<0.01). Although there were no significance in comparison of ARG-1 in two groups, the relative expression of i NOs decreased in U0126 group(P<0.05). After culturing in culture medium,apoptotic speed of MDSCs in U0126 group was faster. The expression of BCL-2 was significantly decreased in U0126 group.Conclusion:(1) IL-17 inhibits apoptosis of MDSCs through ERK1/2.(2) IL-17 promotes tumor growth by inhibiting MDSCs apoposis and enhancing suppressive activity of MDSCs. The effect was associated with apoptosis of MDSCs. |
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