Anti-metastasis Role Of The Antimicrobial Peptides Rich Containing Leu/Lys Residues By Inhibiting Polarization Of Tumor-associated Macrophages Intervention In Breast Cancer

Breast cancer is among the most common malignant tumors worldwide in women.and metastasis is difficult in clinical treatment,which seriously affects the prognosis of patients.At present,there is no effective method for solving breast tumor metastasis.The most frequently found immune cells within the tumor microenvironment are tumor-associated macrophages(TAMs).TAMs are mainly derived from monocytes in the blood circulation that are recruited into the tumor microenvironment,resulting in the differentiation of M2-type macrophages.TAMs promote tumor growth and development and enhance tumor invasion and metastasis.TAM cells.So,it is possible that developing the novel drugs to inhibit M2-type polarization of macrophages may represent a new TAM-targeted approach for angiogenesis,invasion,and metastasis of tumor cells.The antimicrobial peptides rich containing Leu/Lys residues L-K6,L-K5 and L-K6V1 are designed and synthetized in our lab based on the sequence of temporin-1CEb,a peptide isolated fromthe skin secretion of Rana chensinensis.The previous studies showed that the three peptides are able to modulate the polarization process of mouse macrophages RAW264.7,and inhibit growth and proliferation of breast cancer cells.Therefore,in this study,we investigated the effects of the antimicrobial peptides rich containing Leu/Lys residues on the forming of TAMs in tumor microenvironment,and on the metastasis of breast cancer cells induced by TAMs.The human macrophage U937 was selected in this study.Firstly,U937 cells were induced with PMA at a final concentration of 100 ng/mL for 48 hours,and then incubated with IL-4 at a final concentration of 10 ng/mL for 24 hours.After incubation,U937 cells changed from circular-shape into spindle-shape with pseudopodia extension,and from suspension growth to adherent growth.Meanwhile,a large amount of M2 macrophage-specific factors IL-10 and TGF-β1 was produced from the induced-macrophage,and M2 macrophage-specific marker CD206 expressed.Secondly,the PMA-induced U937 cells were treated with the antimicrobial peptides L-K6,L-K5 and L-K6V1 at a concentration that did not affect cell growth(cell viability is more than 80%)and M2 macrophage inducer IL-4 for 24 h.ELISA results showed that the production of M2-type specific factors IL-10 and Arg-1reduced 38.2% to 61% and 82.1% to 89.7% in PMA and IL-4-induced M2 type U937 cells treated with L-K6,L-K5 and L-K6V1,respectively.Our results suggested that the antimicrobial peptides rich containing Leu/Lys residues were able to inhibit the polarization of human macrophages.The expression of the related signal proteins PPARγ、KLF4 and STAT6 was detected by using Western Blotting assay.The results showed that L-K6,L-K5 and L-K6V1 significantly inhibited the expression of PPARγand KLF4,and phosphorylation of STAT6.Among of the peptides,L-K5 at a concentration of 20μM exhibited the highest inhibitory effects on the expression of PPARγ、KLF4 and STAT6 phosphorylation(70.6%、82% and 86.5%).Cell scratch assay and real-time cell migration analysis(RTCA)were used to investigate the anti-metastasis effects of the antimicrobial peptides rich containing Leu/Lys residues on MCF-7 cell in vitro.The results showed that the supernatant of M2-type TAM cells treated with the peptides and IL-4 could inhibit the mobility of MCF-7 cells,and the inhibitory rate was in a concentration(peptide)-dependent manner.ELISA results exhibited that the peptides reduced the production of TGF-β1and EGF,the EMT-promoting cytokines in M2-type TAM cells.L-K5 at a concentration of 20μM exhibited the highest inhibitory effects on the expression of TGF-β1 and EGF(78% and 88%).Western Blotting assay and RT-qPCR were used to detect the expression of proteins and downstream transcription factors related signaling pathway of tumor metastasis.The results showed that the peptides inhibited the expression of mTOR,smad2,and ERK phosphorylated proteins,the signal proteins related tumor metastasis.L-K5 at a concentration of 20μM exhibited the highest inhibitory effects on the expression of the three signal proteins(73%、95% and91.2%).Meanwhile,the peptides inhibited the gene expression of snail and twist.The expression of the marker protein E-cadherin and the interstitial marker protein Vimentin was inhibited,suggested that the antimicrobial peptides rich containing Leu/Lys residues could inhibit the migration of breast cancer MCF-7 cells.After establishing the BALB/c Nude nude mouse tumor model,the survival and body weight of the tumor-bearing nude mice were recorded.The results showed that the antimicrobial peptide rich containing Leu/Lys L-K5 was not only capable of killing tumor-bearing nude mice,but also tumor-bearing.The weight of nude mice also had no significant effect.The volume of breast in situ tumors was measured daily.The results showed that the volume of breast in situ tumors in the L-K5 group was smaller than that in the control group,and the inhibition rates were 21.44%,23.44%,and 32.6% at concentrations of 2.5,5,and 10 mg/kg,respectively.By observing the number of metastasis points on the liver surface,the results showed that the number of metastasis points in the L-K5 group was less than that in the control group,and the inhibition rates were 18.69%,26.17%,and 46.73% at concentrations of 2.5,5,and 10mg/kg,respectively..The results of hematoxylin and eosin staining showed that the area of liver metastases in the L-K5 group was smaller than that in the control group and was associated with L-K5 concentration.It was pointed out by ELISA that L-K5 can reduce the release of IL-10 and Arg-1 in serum.The inhibitory rates of IL-10 release were 14.14%,24.42% and 29.2% at concentrations of 2.5,5 and 10 mg/kg,respectively.The inhibition rate of Arg-1 release was 46.62%,60.36% and 63.7%,which was correlated with L-K5 concentration,suggesting that the therapeutic effect of L-K5 on liver metastasis of breast cancer may be inhibited.Macrophage polarization is an M2 type that promotes tumor development.In conclusion,our results showed that the antimicrobial peptide rich-containing Leu/Lys residues could inhibits M2-type polarization of macrophages through STAT6-related signaling pathways in IL-4-induced human macrophages,resulting in the reduction of production of EMT-promoting cytokine secreted by M2 macrophages,and migration inhibition of breast cancer cells.The antimicrobial peptides rich containing Leu/Lys residues L-K5 has a certain inhibitory effect on the growth and liver metastasis of breast in situ tumors,possibly by inhibiting the polarization of macrophages to promote tumor progression.