Ginsenoside Rh2 Interferes With ERp5,a SMICA-Related Protease,to Promote Tumor Immune Surveillance Of NK Cells

[Background and Purpose]The diagnosis and treatment of breast cancer has been gaining increasing attention.Breast cancer became the most common cancer worldwide in 2020.According to the traditional herbal theory,the deficiency of vitality and the imbalance of yin and yang of organs are the inherent foundations of breast cancer.As an important traditional Chinese medicine for strengthening and tonifying deficiency,ginseng has been widely used in the prevention and treatment of breast cancer over two thousand years.Ginsenoside,the most important active ingredient in ginseng,has been widely studied in breast cancer treatment,but the mechanisms remain unclear.In particular,the effects of ginsenoside on immune regulation only stay in the stage of basic efficacy verification.As the only type of cytotoxic cells in the innate immune system,NK cells can respond quickly to abnormalities in the body,and are the initial force to deal with tumor cells.The function of NK cells is related to the recognition and binding of NKG2D and its related ligand(NKG2DLs).MICA has been regarded as a major human NKG2DLs.Tumor cells will destroy the NKG2D-MICA axis by shedding MICA to form soluble MICA(sMICA),inhibiting the activation of NK cell function and achieving immune escape under the supervision of NK cells.The shedding of MICA requires several proteases,among which ERp5 can comb the protein domain of MICA and expose the cleavage site,which plays a pioneering role in the process of MICA shedding.Based on the previous studies and literature review,Rh2 was used as the representative component of ginsenosides to clarify the inhibition effect of Rh2 on breast cancer,and it was shown that the mechanisms of inhibiting breast cancer by Rh2 was related to the promotion of NK cell activity.We further explored the relationship between Rh2 and MICA exfoliation as well as its key protease ERp5,to enrich the mechanisms of ginsenosides against breast cancer.[Research Contents and Results]In this study,ginsenoside Rh2,was selected for breast cancer research.In light of in vivo experiments,we established MDA-MB-231 in situ and hematogenous metastasis mouse models and 4T1 hematogenous metastasis mouse model with MICA-overexpressing plasmid,and MDA-MB-231 and MCF-7 breast cancer cells were used to construct a co-culture system with NK92MI in vitro to verify the efficacy of Rh2 and explore the underlying mechanisms.The tumor volume and BrdU immunohistochemical results of in situ mouse model mice showed that Rh2 could inhibit the proliferation of MDA-MB-231 breast cancer cells,and in vitro CCK8 experiment also confirmed that Rh2 could inhibit the proliferation of MDA-MB-231,MCF-7 and MICA-4T1 breast cancer cells.In two hematogenous metastasis models,CT scanning and H&E staining showed that Rh2 could significantly inhibit lung metastasis of breast cancer.On the basis of these preliminary results,we further explored the underlying mechanisms.The LDH cytotoxicity assay was used to detect the mortality of breast cancer cells in both in vitro co-culture systems.The results demonstrated that Rh2 could promote the cytotoxicity of NK cells.NK cells were separated from mouse spleen by magnetic beads,and the number of CD107a+NK cells was detected by flow cytometry.NK cells were cultured in vitro and co-incubated with MICA-4T1 cells.The results of both experiments were consistent with the previous findings.In addition,ELISA data of mouse serum revealed that Rh2 could promote the release of perforin,granzyme B and interferon-y,which was also in line with the previous conclusion.Furthermore,we determined the effects of Rh2 on the NKG2D-MICA axis.The NKG2D neutralization assay and western blot analysis showed that the effect of Rh2 on promoting the activity of NK cells was not related to the expression of NKG2D,but depended on the NKG2D-NKG2DLs axis.It is suggested that the enhancement of NK cell activity by Rh2 may be associated with the improvement of MICA expression.In connection with the exfoliation of MICA and the key role of ERp5,western blot and immunofluorescence analysis confirmed the expression of MICA and ERp5 in tumors and lung tissues of mice and the content of sMICA in serum was detected by ELISA.The results showed that Rh2 could inhibit the expression of ERp5,up-regulate the expression of MICA and reduce the formation of sMICA.The similar experiments were performed on MDA-MB-231 and MCF-7 cells in vitro,and the results were consistent with the previous results.In addition,the CETSA data also confirmed that Rh2 and ERp5 in MDA-MB-231 cells exhibited strong binding capacity.[Conclusion and Significance]Based on the above results,it was demonstrated that ginsenoside Rh2 could exert striking effects on inhibiting breast cancer by enhancing the activity of NK cells.Rh2 inhibited the expression of ERp5,interfered with the shedding process of MICA,reduced the production of sMICA,and maintained the expression level of MICA on breast cancer cells.This was essential for NKG2D to activate the killing pathway more efficiently by recognizing MICA,interfering with the immune escape of breast cancer cells,and promoting the immune surveillance function of NK cells.This study illustrated that ginsenoside Rh2 could promote the activity of NK cells by interfering with the immune escape pathway of exfoliated MICA in breast cancer cells,so as to exert an inhibition effect on breast cancer,which complemented the immune regulation mechanisms of ginsenoside against breast cancer,enriched the research of ginsenoside against breast cancer,provided theoretical and experimental basis for the use of ginseng to improve the immunity of patients with breast cancer,and provided new perspectives for the prevention and treatment of breast cancer.