Therapeutic Effects Of Astragalus Extract Combined With5-Fu/CDDP, And The Role Of Interleukin (IL)-6/STAT3Signaling Pathway In Gastric Cancer

Part Ⅰ Inhibitory effects and possible mechanisms of astragalusextract combined with5-Fu/CDDP on human gastric cancerSGC-7901in vitro and on xenograft tumor of nude mice invivoObjective: The objective is to determine the effects of astragalus extract (AE) and itscombination with5-fluorouracil (5-FU)/CDDP on the proliferation and apoptosis of human gastric cancer SGC-7901cells in vitro, and on tumor growth andproliferation in orthotopic transplantation gastric tumor model in nude mice in vivo.The underlying mechanisms involving in IL-6/JAK/STAT3pathway were alsoinvestigated in vivo after AE treatments.Methods: The cell proliferation inhibition rate was detected by MTT assay. The cellapoptosis was observed by the Hoechst33258staining and flow cytometry. Animalmodel of transplanted tumor with SGC-7901was established. Once the tumor grewto the size of100~300mm3, the mice were randomized into7groups: control,5-FU(50mg/kg), CDDP (4mg/kg), AE+5-FU (6g/kg+50mg/kg), AE+CDDP (6g/kg+4mg/kg), AE-low-dose group (3g/kg) and AE-high-dose group (6g/kg). Each groupwas composed of five nude mice and then mice continuously received abovetreatments through oral gavage daily for3weeks. The tumor volume and growth innude mice were measured after3weeks of treatments. The serum levels of IL-6andVEGF were detected by ELISA. Expressions of IL-6, STAT3/p-STAT3, VEGF,Survivin, Bax, and Bcl-2proteins were detected by immunohistochemical stainingand western blot analysis.Results: Treatments with AE,5-Fu, and CDDP alone inhibited the growth of the humangastric cancer cell line SGC-7901cells. Compared with AE,5-Fu, or CDDP alone,combination of AE with5-Fu/CDDP caused higher cell growth inhibition rate (P＜0.01). Treatments with AE and5-Fu, CDDP alone induced the apoptosis ofSGC-7901cells. Combination of AE with5-Fu/CDDP further increased theapoptosis rate of SGC-7901cells as compared with AE,5-Fu, or CDDP alone.Inhibition on the tumor growth was observed after treatment with5-FU, CDDP, andtheir combination with AE. The weight loss in mice treated with5-FU, CDDP, andtheir combination is significantly decreased compared with those in blank control group (P<0.05or P＜0.01). However, the tumor inhibitory effects between los-doseand high-dose of AE were not statistically different. The serum levels of IL-6andVEGF were decreased in the treatment groups, especially in AE plus5-FU/CDDPgroups. Furthermore, treatments with AE and its combination with5-FU/CDDPdecreased the expression of IL-6, total and phosphorylated STAT3, nuclear level ofphosphorylated STAT3, Survivin, VEGF, and Bcl-2proteins, whereas theexpression of Bax was increased after AE treatment in tumor tissues of nude micetransplanted with human gastric cancer cells.Conclusion: Combination of AE with5-Fu/CDDP inhibited cell growth and inducedapoptosis of human gastric cancer SGC-7901cells as compared to treatment withAE,5-FU, or CDDP alone. In addition, treatment with AE plus5-Fu/CDDPdecreased tumor growth in nude mice transplanted with human gastric cancer cells.The mechanisms underlying these findings are associated with decreased activationof IL-6/STAT3signals and augmented apoptosis after treatment with AE and/or5-Fu/CDDP. Part Ⅱ Clinical implications of interleukin (IL)-6-typecytokines/STAT3signal pathway in human gastric cancer,and immnoregulational effects of astragalus extract inpatients with gastric cancerObjective: Signal transducers and activators of transcription3(STAT3) signal pathwayplays important roles in the pathogenesis of gastric cancer. In the present study, wefirst measured the level and expression of key proteins in interleukin6(IL-6)/STAT3signal pathway in serum and gastric cancer tissue. Then thecorrelation between the STAT3activation in local tumor tissue and clinicalimplication was analysed. Lastly, the change of immune function after operation andthe immnuoregulative effects of astragalus extract (AE) were determined in patientswith gastric cancer.Methods: Total71fresh gastric cancer tissues and30normal gastric tissues (5cm faraway from the tumor) were obtained from gastric cancer patients who underwentgastrectomy in the Division of General Surgery, the First Affiliated Hospital ofAnhui Medical University, P.R. China, from Feb.2010to Jun.2012. Blood sampleswere collected before operation,1week,1month after surgical gastrectomy, andduring the chemotherapy to measure the levels of IL-6, IL-10and vascularendothelial growth factor (VEGF) using an enzyme-linked immunosorbent assay(ELISA), as well as T lymphocyte subsets (CD3+, CD4+, CD8+, CD4+/CD8+) andnatural killer (NK) by a flow cytometry. Furthermore, the expression of IL-6,survivin, STAT3, STAT3phosphorylation (p-STAT3), and VEGF were determinedin human gastric cancer and adjacent normal mucosa through Western blot andimmunohistochemistry. Results: Serum IL-6, IL-10and VEGF levels were higher before operation, and thendecreased at1week,1month after surgery. Western blot and immunohistochemistrystaining showed that STAT3activation was correlated with IL-6expression.Furthermore, the activation of STAT3resulted in upregulaton of its downstreamgenes including VEGF and Survivin. The percentages of T-cell subsets (CD3+, CD4+,and CD4+/CD8+ratio) and NK cells in blood were significantly increased atpostoperative-week1as compared to those in preoperative group, which was furtheraugmented at1month after gastrectomy. Treated with AE during the chemotherapyincreased the percentages of T-cell subsets (CD3+, CD4+, and CD4+/CD8+ratio) andNK cells in the blood of patients with gastric cancer.Conclusion: Increased IL-6-induced activation of STAT3was observed in neoplasticgastric tissue, which postively correlated with the increased expression of itsdownstream genes such as VEGF and Survivin. Moreover, the T-cell subsets andNK cells with anti-tumor property in serum play an important role in thepathogenesis of gastric cancer. AE treatment is beneficial to the gastric cancerpatients through its immnoregulational effects.