Effect Of Plumbagin On Angiogenesis Of Gastric Cancer And Its Mechanism

Gastric cancer is a worldwide malignant tumor.In 2020,the incidence rate of gastric cancer was fifth in the world,about 1 million and 89 thousand new cases one year.Although there are many treatments for gastric cancer,including surgical resection,neoadjuvant chemotherapy,radiotherapy,molecular targeted therapy and immunotherapy,the 5-year overall survival rate of gastric cancer patients is only 35.1%.Tumor angiogenesis is an important link in the development of tumor,which supports the growth and metastasis of tumor.Therefore,inhibiting angiogenesis is a promising strategy for anticancer drugs.Plumbaginones belong to 1,4-naphthoquinones,which are important quinones.At present,studies have shown that plumbagin has good anticancer effect in liver cancer,breast cancer and prostate cancer.Angiogenesis is a new blood vessel generated by the original blood vessels,which plays an important role in tumor growth and metastasis.It has been reported that plumbaginone can induce the expression of VEGF receptor 2(VEGFR2)and inhibit the migration and invasion of tumor endothelial cells.Although plumbaginone has been reported in anticancer research,its anticancer effect and mechanism on gastric cancer have been reported.Many studies have shown that the invasiveness of gastric cancer is related to the activity of nuclear factor kappa B(NF-?b)transcription factor.NF-?B pathway is an important signal pathway to improve the body's inflammatory response,as well as the survival,apoptosis and proliferation of cancer cells.Therefore,it is of great practical significance to select NF-? B pathway to explore the anticancer effect of plumbaginone on gastric cancer and the mechanism of inhibiting its angiogenesis.In this study,the effects of plumbagin on the proliferation,migration and anti angiogenesis of gastric cancer cells were studied by gastric cancer cell,HUVECs cell experiment and gastric cancer mouse model animal experiment.The mechanism of plumbagin inhibiting angiogenesis of gastric cancer was analyzed by TNF-?mediated NF-? B pathway,which provided a theoretical basis and reference for the development of drugs for the treatment of gastric cancer.Part I Effect of Plumbagin on angiogenesis of gastric cancerObjectiveIn this part of study,cell experiment was used to analyze the effects of different doses of plumbaginone on the proliferation and angiogenesis of gastric cancer cells by analyzing the proliferation and migration ability of gastric cancer cells and the luminal structure formation ability of HUVECs.MethodThe proliferation of gastric cancer cells and endothelial cells before and after treatment with plumbaginone was analyzed by enzyme-linked immunosorbent assay.The migration ability of gastric cancer cells and endothelial cells was analyzed by cell scratch test.HUVECs were cultured with Matrigel and the effect of plumbaginone on the formation of lumen structure was analyzed by microstructure.Meanwhile,real-time fluorescent quantitative PCR and Western blot were used The mRNA and protein levels of VEGF and VEGFR2 were detected by blot.Results1.The number of proliferation of gastric cancer cells was lower than that of the blank control group,and with the increase of the dose of Plumbagin,the number of cell proliferation decreased significantly(P<0.05).At the same time,compared with the blank group,the proliferation of HUVEC cells was significantly inhibited by different doses of Plumbagin treatment for 24 hours(P<0.01).and with the increase of Plumbagin dose,the number of cell increment gradually decreased(Figure 1.2),which shows that Plumbagin can inhibit the proliferation of HUVEC cells.The number of HUVEC cells in the same dose group was significantly lower than that of SGC-7901 cells,which indicated that the inhibitory effect of Plumbagin on the proliferation of vascular endothelial cells was stronger than that of gastric cancer cells.It is suggested that Plumbagin can inhibit the proliferation of gastric cancer cells and HUVEC cells.2.24 hours after the gastric cancer cells were scratched,we can see that the scratch repair ability of different doses of Plumbagin treatment group was significantly weaker than that of the blank control group,and with the increase of Plumbagin treatment dose,the scratch repair ability gradually weakened.At 24h after the scratch of HUVEC cells,the scratch repair ability of the treatment group with different doses of Plumbagin was significantly weaker than that of the control group,and the trend was similar to that of gastric cancer cells.At the same time,compared with the blank control group,the migration ability of HUVEC cells and gastric cancer cells decreased significantly with different doses of Plumbagin,and the migration distance was shorter with the increase of dose,but HUVEC cells were far from gastric cancer cells as a whole.It is suggested that Plumbagin can inhibit the migration of gastric cancer cells and HUVEC cells.3.HUVECs in the control group can form a cross-linked vascular network structure,while HUVEC cells can't form a complete lumen structure after treatment with Plumbagin,and some individual cord like structures will not appear.At the same time,the formation of lumen structure in the high dose group was significantly lower than that in the low dose group.It is suggested that Plumbagin has antiangiogenic effect.4.In SGC-7901 cells,the mRNA expression levels of VEGF and VEGFR2 were down regulated by 2 ? m,4 ?m and 6 ?m plumbaginone,and the down-regulation effect was significant with the increase of plumbaginone treatment dose(P<0.05).Western blot results showed that the protein expression level was consistent with the mRNA expression level.These results suggest that Plumbagin can inhibit the expression of angiogenesis related factors in gastric cancer cells.5.In HUVEC cells,the mRNA expression levels of VEGF and VEGFR2 were down regulated by 2 ? m,4 ? m and 6 ? m plumbaginone,and the down-regulation effect was significant with the increase of plumbaginone treatment dose(P<0.05).Western blot results showed that the protein expression level was consistent with the mRNA expression level.All of these indicated that Plumbagin could inhibit the expression of angiogenesis related factors in HUVEC cells.Conclusion1.Plumbagin can inhibit the proliferation and migration of gastric cancer cells.2.Plumbagin can inhibit the angiogenesis of HUVEC cells.3.Plumbagin can down regulate the expression of VEGF and VEGFR-2.Part ?:Effect of Plumbagin on angiogenesis of gastric cancer xenografts in nude miceObjectiveIn this study,gastric cancer cells were used to induce the establishment of gastric cancer model in mice.The inhibitory effects of plumbaginone on the growth and angiogenesis of gastric cancer were studied via constructing subcutaneous transgraft tumor model.And macro analysis,ELISA and immunohistochemistry were performed.MethodsSixty BALB/c mice were selected to establish gastric cancer model by subcutaneous injection of gastric cancer cell SGC-7901,and randomly divided into four groups,model group,low-dose plumbagin group(2 mg/kg),medium-dose plumbagin group(4 mg/kg)and high-dose plumbagin group(6 mg/kg).The tumors were removed after death,the volume and weight of tumors were measured,and the ratio of anti-tumor was analyzed.The contents of VEGF.VEGFR2 in serum and tumor tissues were tested by ELISA and immunohistochemical staining.The microvessel density(MVD)was evaluated by immunohistochemistry.Results1.Compared with the model group,the tumor size and weight of mice treated with plumbaginone decreased significantly,and with the increase of dosage,the tumor size and weight decreased gradually,indicating that plumbaginone had significant inhibitory effect on gastric cancer(P<0.05).The inhibition rate of high dose group(6mg/kg)was 50.32%.2.ELISA results showed that compared with the model group,the serum VEGF and VEGFR2 levels of different doses of plumbagin were significantly decreased(P<0.05),and the expression levels of VEGF and VEGFR2 gradually decreased with the increase of dosage.These results indicate that plumbagine can effectively reduce the levels of VEGF and VEGFR2 in serum.3.Immunohistochemical staining and microscopic observation showed that the positive expression of CD31 was the most obvious in the model group,and the number of brown tubes was the most in the model group;the positive expression of plumbagin in low,medium and high dose groups showed a gradual decrease trend,which indicated that plumbagin could effectively inhibit the angiogenesis of transplanted tumor in nude mice.4.The results of electron microscopy showed that VEGF and VEGFR2 were mainly expressed in the cells,and the positive expression of VEGF and VEGFR2 in the model group was the most significant,while the positive expression of VEGF and VEGFR2 in low,medium and high dose groups was gradually decreased(P<0.05).These results demonstrated that plumbaginone can inhibit the proliferation and angiogenesis of vascular endothelial cells through upregulating VEGF and VEGFR2.5.Western blot results showed that compared with model group,the levels of NF-kB p-p65/NF-?B-p65,p-IKK/IKK and p-IKB?/IKB? were evidently decreased in medium and high dose of plumbagin groups(p<0.05).These proteins were expressed in a dose-dependent manner.Conclusion1.Plumbagin could inhibit the growth of gastric cancer in nude mice.2.Plumbagin can prevent the gastric cancer from creating blood vessels.of Part III:Mechanism of Plumbagin in inhibiting angiogenesis of gastric cancerObjectiveIn this study,we selected NF-?B signaling pathway and analyzed the regulatory effect of plumbagin on angiogenesis of gastric cancer through the protein expression of NF-?B p65,phosphorylated IKK and I ? B ? in the pathway,and confirmed the relationship between NF-? B signaling pathway and angiogenesis of gastric cancer by combining with immunohistochemical analysis of human gastric cancer tissue.MethodHUVEC cells mediated by TNF-? were co cultured with Plumbagin,and the formation of lumen structure was analyzed by Matrigel.The expression of NF-? B p65,phosphorylated IKK and IKB ? in HUVEC was analyzed by Western blot.Meanwhile,the expression of NF-? B pathway related signal molecules and VEGF and VEGFR-2 were analyzed by immunohistochemistry.Results1.The microscopic structure showed that HUVECs in the control group could form cross-linked vascular network structure.while HUVEC cells treated with plumbaginone could not form a complete lumen structure,and some individual cord like structures could not appear,indicating that plumbaginone could affect the formation of vascular lumen by inhibiting HUVEC.2.Western blot results showed that compared with the control group,the protein expressions of NF-? B p65,phosphorylated IKK and I ? B ? were significantly down regulated in plumbagin intervention group(P<0.05).These results indicated that plumbagin could inhibit NF-? B expression by inhibiting NF-? B expression The activation of p65 and phosphorylation of IKK and I ? B ? can effectively block the NF-? B pathway mediated by TNF-?,and then inhibit the ability of endothelial cells to form neovascular lumen.3.The expression levels of NF-? B p65,phosphorylated IKK and I ? B ?,VEGF and VEGFR-2 in gastric cancer tissues were higher than those in adjacent normal tissues.Combined with our previous research results,it is suggested that NF-? B pathway may be a therapeutic target of plumbaginone in inhibiting gastric cancer in human body,and through their down regulation,it is clear that plumbaginone has the potential of anti gastric cancer vascular effect in clinical application.Conclusion1.Plumbagin blocks NF-? B signaling pathway by inhibiting TNF-? mediated activation of NF-? B p65,phosphorylation of IKK and I ? B ? expression,thus affecting the ability of endothelial cells to form vascular lumen.2.NF-? B pathway related factors(including NF-? B p65,phosphorylated IKK and I ? B ?)and angiogenic factors VEGF and VEGFR-2 were highly expressed in patients with gastric cancer.Combined with the previous conclusion,it is suggested that this pathway may be an effective target for plumbagine to inhibit gastric cancer in human body.