The Anti-tumor Mechanism Of Active Compounds In Xiao’ai Jiedu Prescription

Background and objectives:Xiao’ai Jiedu Prescription is an effective prescription created by professor Zhou Zhong-Ying based on his years of clinical experience in cancer treatment.Previous clinical and experimental studies in professor Wu Mian-Hua’s research group have confirmed that Xiao’ai Jiedu Prescription can effectively treat a variety of malignant tumors.However,it has been largely unknown which bioactive components,and especially how the combination of these bioactive components,may contribute to the effective anti-cancer activity of the prescription.The objectives of this study were to evaluate the effect of 2-hydroxy-3-methylanthraquinone(HMA),a bioactive component in Hedyotis diffusa Willd on lung cancer cells,to observe the anti-cancer effects of the extraction of Xiao’ai Jiedu Prescription,to determine the anti-glioma effects of active compounds in Xiao’ai Jiedu Prescription,and to investigate the potential synergistic effect between active compounds on glioblastoma cells aiming to explore the material basis in the prescription.Methods:Viability of cancer cells was determined by CCK-8 assay or MTS assay.Cell survival was determined by colony formation assay.Apoptosis was detected by Annexin V-FITC/PI flow cytometric analysis.Cell cycle distribution was detected by flow cytometry.Migration and invasion abilities of cells were determined by wound-healing assay and transwell invasion assay,respectively.Sphere formation assay was applied to evaluate self-renewal ability of cancer stem cells.Protein array assays were applied for multiplex measurement of pathway-related biomarkers.Gene expression and protein levels of related molecular markers were quantitated by real time-PCR and Western blot analysis,respectively.Results:HMA significantly inhibited IL-6-stimulated growth and colony formation of A549 cells,increased the number of apoptotic cells,and inhibited A549 cell invasion associated with downregulation of expression of IL-6-induced MMP-1,MMP-2,and MMP-9 genes.IL-6 increased the levels of tyrosine phosphorylation of JAK2 and STAT3 in A549 cells,which was reversed by HMA treatment.In addition,HMA reduced the expression of a series of inflammation-related cytokines in A549cells supernatant,including IL-6,G-CSF,IL-6R,1L-8,MCP-1，RANTES,TNF-a.Water extraction,ethanol hot extraction,ethanol cold extraction,butanol extraction of Xiao’ai Jiedu Prescription showed certain anti-proliferative activities against lung cancer,glioma,breast cancer,and colon cancer,with ethanol extraction and butanol extraction more active than water extraction.Glioma cells were more sensitive than others to the treatments.We then evaluated the anti-glioma cancer cell activities of 12 major compounds in the Xiao’ai Jiedu Prescription,including Ergosterol,Hederagenin,Kaempferol,Ursolicacid,Oleanolic acid,Stigmasterol,2-hydroxy-3-methylanthraquinone(HMA),Ginsenoside Rg3,Ophiopogonin B(OPB),Liriope muscari baily saponins C(DT-13),Ophiopogonin D(OPD),and Ophiopogonin D’(OPD’).These compounds could significantly inhibit the growth of U118,LN229,SW1783,SW1088 human glioma cells.Among them,UA,OPB,DT-13 and OPD’ showed the most significant effect.To further understand the principle of Xiao’ai Jiedu Prescription,we evaluated the combination effects of bioactive compounds to determine if any combination might have synergistic effect.Ursolicacid(UA)and Ophiopogonin D1(OPD’),the active components in Hedyotis diffusa Willd and Ophiopogon japonicas respectively,significantly inhibited the growth of U118,LN229,SW1783,SW1088 human glioma cells dose-dependently,and LN229 cells were more sensitive than others to the treatments.The combination of UA and OPD’significantly inhibited the growth,survival,migration,invasion,and cancer stem cell self-renewal of LN229 cells in a synergistic manner.Further studies showed that the combination of UA and OPD’ could act synergistically to increase cell apoptosis.The apoptosis-specific protein array assay showed that the combination of UA and OPD’ could increase the protein level of Catalase and decrease the protein level of Survivin,Claspin,HO-1,and HTRA2 in a synergistic manner.Conclusions:The results of our studies indicate that HMA may inhibit the growth and invasion of lung cancer cells in part via downregulation of IL-6-induced JAK2/STAT3 pathway,and that UA and OPD’,especially their synergistic combinations,may have potent activities against glioblastoma in part via induction of apoptosis in a synergistic manner.Our results also elucidate the anticancer mechanism of Xiao’ai Jiedu Prescription and provided experimental evidence to support the scientific basis underlying Xiao’ai Jiedu Prescription.