The Mechanism Of Pterostilbene Inhibiting The Growth And Invasion Of Mice Transplanted Breast Tumor And Remodeling Its Tumor Microenvironment

With the increasing of treatment methods,the mortality rate of breast cancer has been decreasing day by day,but the incidence rate is increasing due to various factors.Finding safe and effective methods to reduce the incidence of breast cancer and inhibit the growth and metastasis of breast cancer has become a research hotspot.Pterostilbene(PTE)is a natural compound found in berries and a derivative of resveratrol.Studies have shown that PTE not only has higher bioavailability,longer half-life,lower toxicity and higher safety than resveratrol,but also has has the function of preventing tumor and anti-tumor.Pyroptosis is an inflammatory cell death in which a family of gasdermin(GSDM)proteins are cleaved by different proteases,causing them to punch holes in cell membranes,which releases intracellular pro-inflammatory factors and damage-associated Molecular Patterns(DAMPs),triggering a strong immune response.Pyroptosis has been found to be involved in various disease processes and its role in the development of tumor has attracted increasing attention.In this paper,above all,we studied the inhibitory effect of PTE on EMT6 and 4T1 breast tumor cells in vitro,and discussed the mechanism of PTE.Subsequently,the EMT6 breast tumor transplated mice model was established and combined with the RNA-seq data to explore the effects of pretreatment and treatment of PTE on the growth of transplanted tumor,immune organs and tumor microenvironment.The main results are as follows:(1)The inhibitory effect of PTE on EMT6 and 4T1 cells and its pyroptosis pathway were studied in vitro.The results of CCK-8,Wound healing and Transwell showed that PTE inhibited the activity of EMT6 and 4T1 cells in a time and concentration-dependent manner.PTE had a concentration-dependent inhibitory effect on migration and invasion of EMT6 and4T1 cells.These results suggest that PTE can effectively inhibit the proliferation,migration and invasion of mouse breast tumor cells.Morphological observation showed that PTE induced mouse breast cancer cells to become round and swollen,and large bubbles appeared in plasma membrane,and the number of deformed cells was proportional to the concentration of PTE,and this deformation was consistent with the characteristics of cell pyroptosis.The results of transmission electron microscopy,LDH release rate and flow cytometry further confirmed that PTE can cause cell membrane rupture of EMT6 and 4T1 cells and induce pyroptosis of cancer cells.The results of q PCR showed that PTE significantly increased the m RNA expression levels of pyroptosis related factors(Caspase-1,GSDMD,BAX,Caspase-9,Caspase-3,GSDME,IL-1β,IL-18,NLRP3 and HMGB1)in two kinds of breast tumor cells.The expression level of pyroptosis related proteins were further detected by WB.The results showed that the protein expression levels of BAX,Cleaved caspase-3 and GSDME-N were significantly increased in EMT6 cells following treatment with PTE,suggesting that PTE may promote pyroptosis of EMT6 cells through the Bax-Caspase-3-GSDME pathway.The protein expression levels of GZMB,BAX,Cleaved Caspase-3 and GSDME-N were significantly increased in 4T1 cells following treatment with PTE,suggesting that PTE may promote pyroptosis of 4T1 cells through the GZMB-Bax-Caspase-3-GSDME pathway.(2)The in vivo study was conducted to study the preventive and therapeutic effects of PTE on the EMT6 breast cancer transplanted tumor.80 healthy female BABL/C mice weighing 18-22 g and aged 6-8 weeks were randomly divided into 8 groups:control group(CON),model group(MOD),PTE pre-treatment low-dose group(PRE-PTE L,80 mg/kg),PTE pre-treatment middle-dose group(PRE-PTE M,200 mg/kg),PTE pre-treatment high-dose group(PRE-PTE H,400 mg/kg),PTE treatment low-dose group(PTE L,80mg/kg),PTE treatment middle-dose group(PTE M,200 mg/kg),and PTE treatment high-dose group(PTE H,400 mg/kg),with 10 mice in each group.After gavaging the PRE-PTE group according to different dose for 14 d,the CON group was inoculated with 0.1m L PBS,and other groups were established the mice model.7 d after tumor inoculation,and the tumor volume in the PRE-PTE group was significantly smaller than that of other tumor-bearing mice in a dose-dependent manner.Measured the tumor volume of tumor-bearing mice 7 d after inoculation.The CON and MOD groups were gavaged with 0.1 m L olive oil per day,and the PRE-PTE and PTE groups were daily gavaged PTE according to different doses for 21 d.Observed the status of mice and plotted the curves of body weight and tumor growth.After treatment,collected transplanted tumor tissues in each group,weighed the tumor mass and calculated the tumor inhibition rate.The results showed that,PTE had no effect on the spirit,appetite and body weight of the tumor-bearing mice.The tumor volume and growth rate of the MOD group were significantly higher than those of the PRE-PTE and PTE groups.The growth rate of tumor volume in the PRE-PTE group and PTE group was gradually accelerated16 days after tumor inoculation.Under the same dose condition,the tumor volume and growth rate of the PRE-PTE group,as well as the weight of in vitro transplanted tumor were significantly smaller than those in the PTE group,and the tumor inhibition rate was significantly higher than that in the PTE group.HE staining was used to observe the pathological changes of transplanted tumor tissues in each group.The results showed that the number of tumor cells and blood vessels in the MOD,PTE and PRE-PTE groups gradually decreased,the arrangement of tumor cells gradually loosened,and the phenomenon of nucleolysis and cell necrosis gradually increased.q PCR and WB were used to detect the m RNA and protein expression levels of pyroptosis related factors in tumor tissues.The results showed that,compared with the MOD group,PTE significantly increased the m RNA expression levels of Caspase-1,GSDMD,BAX,Caspase-9,Caspase-3,GSDME,IL-1β,IL-18,NLRP3 and HMGB1.The protein expression level of BAX,Cleaved Caspase-3,GSDME-N,and the PRE-PTE group were significantly increased,and the PRE-PTE group was more significant than the PTE group.These results indicate that PTE can effectively inhibit the growth of breast cancer transplanted tumor without affecting the spirit,appetite and body weight of mice.At the same dose,the PRE-PTE group showed better tumor inhibition than the PTE group.This inhibitory effect may be realized through the Bax-Caspase-3-GSDME pathway to promote the pyroptosis of tumor cells and release a large number of inflammatory factors.(3)RNA-seq and data analysis were used to explore the effect of PTE pre-treatment and treatment on gene expression.Three transplanted tumors in each group of the MOD,PTE H and PRE-PTE H group were randomly selected for transcription level detection,and the differential genes between each two groups were analyzed and screened.8 genes were randomly selected for q PCR verification.The results showed that the RNA-seq data were reliable.Functional annotation and pathway enrichment analysis were conducted for the differential genes between each two groups,and the differential genes shared by the three comparison groups were created into a gene set,and clustering,annotation and enrichment analysis were conducted.The results showed that the DEGs were significantly different in the three groups,and were mainly enriched in immune response activation related pathways.The immune system related genes in the common DEGs were constructed as a gene set and analyzed by clustering and enrichment.The results showed that PTE significantly increased the expression of immune system-related genes,especially in the PRE-PTE group.DEGs mainly focus on positive regulation of lymphocyte activation and regulation of T cell activation.These results suggest that PTE pretreatment may play a role in cancer prevention by improving immunity.When cancer occurs,PTE plays an anticancer role through positive modulation of immune response activation.The protein-protein interaction(PPI)map of common DEGs was constructed,and the key targets such as Ptprc,Cd4,Stat1 and Cxcl9 were found,which are related to immune system.The PPI map of immune system related genes was further constructed,and the key targets such as Cd4,Ptprc,Cxcl9 and Cxcl10 were found,which are related to T cell receptor signaling pathway.PPI results were consistent with the above cluster and enrichment analysis results,indicating that immune system related pathways play a crucial role in the tumor inhibition of PTE,laying a foundation for further research on the effect of PTE on tumor immune microenvironment.(4)Flow cytometry,IHC and RNA-seq data were used to explore the effects of pre-treatment and treatment of PTE on tumor microenvironment,and to clarify the mechanism of PTE inhibiting tumor growth.After 21 d of administration,the appearance,weight,index and HE staining of the spleen in vitro were compared.The results showed that tumor bearing could decrease the spleen state,while PTE could improve the spleen state.Randomly selected three mice in each group of the CON,MOD,PTE H and PRE-PTE H group.Collected peripheral blood and obtained single cell suspension of spleen and tumor tissues by grinding method.Used flow cytometry to detect the percentage of immune cells.The results showed that compared with the CON group,tumor-bearing group significantly decreased the percentage of tumor-suppressor immune cells(such as CD4~+T cells,CD8~+T cells and NK cells),and increased the percentage of tumor-promoting immune cells(such as MDSC).Compared with the MOD group,PTE significantly decreased the percentage of tumor-suppressor immune cells and increased the percentage of tumor-promoting immune cells.In tumor tissues,PTE significantly decreased the percentage of M2-TAMs and increased the percentage of M1-TAMs,as well as increased the ratio of M1/M2-TAMs.IHC results showed that PTE significantly increased the percentage of B cells,decreased the percentage of Treg cells,and decreased MVD in tumor tissues.q PCR results showed that PTE significantly increased the m RNA expression levels of immune activating factors(IL-2,IFN-γ,TNF-α,CSF-1 and CCL-2),decreased the m RNA expression levels of immune suppressive factors(IL-4,IL-10 and TGF-β)and angiogenesis related factors(HIF-1αand VEGFA).WB results showed that PTE significantly reduced the protein expression levels of angiogenesis related proteins such as HIF-1αand VEGFA,and invasion and metastasis related proteins such as MMP-2 and MMP-9.Among the above results,the PRE-PTE group was more significant than the PTE group.These results suggest that after the successful establishment of transplated tumor model,PTE could adjust the immune system,increase tumor suppression immune cells and immune promoting factor,decrease tumor promoting immune cells and immune inhibitory factor.In tumor tissues,PTE could adjust the T cells classification,promote the differentiation of CD4~+T cells into Th1 type,inhibit the differentiation of CD4~+T cells into Th2 type,regulate TAMs polarity,promote TAMs to M1-type polarization,inhibit TAMs to M2-type polarization.PTE could alleviate the immune suppression caused by tumor bearing,enhance the killing effect of the immune system on tumor cells,improve the state of spleen,inhibit tumor angiogenesis,invasion and metastasis,thus inhibit tumor growth,and the effect of PTE pretreatment is better.Conclusion:(1)PTE significantly inhibited the proliferation,migration and invasion of EMT6 and 4T1 cells.(2)PTE may promote pyroptosis of EMT6 cells through the BAX-Caspase-3-GSDME pathway,and may promote pyroptosis of 4T1 cells through the GZMB-BAX-Caspase-3-GSDME pathway.(3)PTE can effectively inhibit the growth of transplanted tumor of breast cancer in mice with little toxicity and pretreatment had better inhibitory effect than treatment.This may be achieved by promoting pyroptosis of breast cancer cells through the BAX-Caspase-3-GSDME pathway and releasing a large number of inflammatory factors.(4)PTE pretreatment can improve immunity and inhibit tumor more siginificantly than treatment.It is speculated that the inhibitory effect of PTE on transplanted breast tumor is achieved by promoting pyroptosis of breast tumor cells and releasing inflammatory factors,thus recruiting immune cells,regulating systemic immune function and tumor immune microenvironment,and inhibiting tumor angiogenesis,invasion and metastasis.