Hypoxic Macrophage-derived VEGF Promotes Proliferation And Invasion Of Gastric Cancer Cells

Background Gastric cancer is one of the most common malignant tumors in the world.In recent years,with the improvement of hygienic conditions and the improvement of eating habits,the incidence of gastric cancer has decreased.However,due to the characteristics of high metastasis rate and high recurrence rate of gastric cancer,the prognosis of patients with advanced gastric cancer is still poor,and the average 5-year survival rate is only about 15%.The distribution of tumor cells is the main cause of death and the biggest obstacle to treatment.However,the specific mechanism of metastasis in gastric cancer cell is still unclear.Therefore,gastric cancer has posed a serious threat to human health and brought huge burden to social and economic development.It has aroused widespread concern in the medical community and is a major scientific issue that needs to be solved urgently in modern medicine and health.During tumor development,macrophages are recruited in tumors by hypoxia and molecular factors released by tumors,including growth factors and cytokines.Tumor associated macrophages(TAM)might be converted from a tumor suppressor M1 subtype to a tumor-promoted M2 subtype and promotes angiogenesis and immunosuppression,furthermore,TAM can be locally recruited in cancer by induction of signaling molecules such as chemokines,which would contribute to angiogenesis and increase vascular permeability.TAM can release a variety of biologically active substances under hypoxic conditions,and are widely involved in tumor proliferation,invasion and metastasis,but their biological processes affecting the invasion and metastasis of gastric cancer and related signal transduction pathways are still unclear.Therefore,this study intends to design experiments to explore the effects of activated macrophages under hypoxia on the proliferation and invasion of gastric cancer cells,trying to find out the key regulatory proteins or signal transduction pathways in this biological process,which is conducive to targeted treatment of gastric cancer metastasis and provide precise individualized treatment for gastric cancer patients.Objectives To explore the role of hypoxic macrophages in the process of gastric cancer metastasis cells and there regulation mechanism by studying the effects of hypoxia on macrophages,and to elucidate the effects of active substances released by macrophages on the proliferation and invasion of gastric cancer cells under hypoxia.To study the molecular mechanism of macrophage-associated regulatory mediators to promote the proliferation and invasion of gastric cancer cells,in order to find key proteins or biomarkers in the process of gastric cancer metastasis,and provide new ideas and theoretical basis for early diagnosis,risk warning and precision treatment of gastric cancer metastasis.Materials and methods1.Cell culture and establishment of In vitro hypoxia model of macrophages The human mononuclear macrophage cell line THP-1 and the gastric cancer cell line SGC7901 and MKN45 were cultured in RPMI-1640 medium containing 10%fetal calf serum at a 37 ° C,5% CO2 cell culture incubator.The cultured THP-1 cells were stimulated with phorbol ester(PMA,150 n M)for 24 hours to induce differentiation into macrophages,and then the cells were placed in an anoxic incubator containing 5% carbon dioxide and 95% nitrogen to establish the hypoxia model.2.Collection of macrophage-derived regulatory media After the macrophage was hypoxic for a period of time,the macrophage culture medium(H-CM)under hypoxia was collected in an anoxic operation table,and centrifuged at 4 ° C,1000 rpm for 5 minutes,and centrifuged.The supernatant is transferred to a new EP tube for later use.3.Q-PCR and ELISA Q-PCR and ELISA were used to detect the expression of VEGF m RNA and VEGF protein in macrophages under hypoxic conditions.4.Proliferation and invasion experiment of gastric cancer cell SGC7901 and MKN45The effect of H-CM on the proliferation of SGC7901 and MKN45 cells was observed using CCK-8 cell proliferation assay and Transwell tumor invasion assay.5.Determination of protein BCA protein content The protein concentration in the cell lysate sample was measured using a BCA protein quantification kit.6.Western blot Western blot was used to detect the activation of signal transduction pathways in gastric cancer cells.7.Statistical analysis SPSS 20.0 software was applied for data statistics in our study,and the data were showed as mean ± standard deviation(` x ± s).P < 0.05 was considered statistically significant for all experiments.Results1.Hypoxia promotes the expression of THP-1 in macrophage cells in vitro Q-PCR and ELISA were used to detect the changes of VEGF m RNA levels in macrophages and VEGF protein levels in culture supernatants under hypoxia.The results showed that hypoxia significantly up-regulated VEGF m RNA levels in macrophages and protein levels in culture supernatants,and this up-regulation was time-dependent.The expression of VEGF increased with the prolongation of hypoxia.2.H-CM enhance the proliferation and invasion of SGC7901 and MKN45 gastric cancer cells By using CCK-8 cell proliferation assay and transwell invasion assay,H-CM can significantly promote the proliferation and invasion of SGC7901 cells MKN45 cells,and the proliferation and invasion ability of SGC7901 and MKN45 cells increased with the increase of supernatant concentration.3.Inhibition of VEGF receptor significantly inhibit H-CM-induced proliferation and invasion of gastric cancer cells Pretreatment of SGC7901 and MKN45 cells with the VEGFR receptor-specific inhibitor Nintedanib could significantly inhibit H-CM-induced proliferation and invasion of SGC7901 and MKN45 cells.4.H-CM promotes activation of PI3K/Akt and p38 MAPK signaling pathways in SGC7901 and MKN45 gastric cancer cells Western blot was used to detect the activation level of PI3K/Akt signal transduction pathway by H-CM in SGC7901 and MKN45 cells.The results showed that H-CM significantly enhanced the phosphorylation of Akt and p38 in SGC7901 and MKN45 cells,and H-CM was concentration-dependently activated by PI3K/Akt and p38 MAPK signal transduction pathways.5.Inhibition of VEGFR significantly inhibits H-CM-induced activation of PI3K/Akt and p38 MAPK signaling pathways in SGC7901 and MKN45 gastric cancer cell s Pretreatment of SGC7901 and MKN45 cells with the VEGFR receptor-specific inhibitor Nintedanib can significantly inhibit H-CM-induced activation of PI3K/Akt and p38 MAPK signaling pathways in SGC7901 and MKN45 cells.6.Inhibition of PI3K/Akt and p38 MAPK signal transduction pathway activation can significantly inhibit H-CM-induced proliferation and invasion of SGC7901 and MKN45 cells.SGC7901 and MKN45 cells were pretreated with LY294002,a specific inhibitor of the PI3K/Akt signaling pathway.Or SB203580,a specific inhibitor of the p38 MAPK signaling pathway.The study found that specific inhibitors of PI3K/Akt or p38 MAPK signaling pathways can significantly reduce H-CM-induced proliferation and invasion of gastric cancer cells.Conclusions1.The transcription and expression levels of VEGF are increased in macrophages induced by hypoxia;2.VEGF can affect the proliferation and invasion ability of gastric cancer cells;3.Hypoxia-induced macrophage-derived VEGF may enhance the proliferation and invasion of gastric cancer cells by activating PI3K-Akt and p38 signaling pathways.