title: Astragalus saponins modulates colon cancer development by regulating calpain-mediated glucose-regulated protein expression.
abstract: BACKGROUND
  Glucose-regulated proteins (GRP) are induced in the cancer microenvironment to promote tumor survival, metastasis and drug resistance. AST was obtained from the medicinal plant Astragalus membranaceus, which possesses anti-tumor and pro-apoptotic properties in colon cancer cells and tumor xenograft. The present study aimed to investigate the involvement of GRP in endoplasmic reticulum (ER) stress-mediated apoptosis during colon cancer development, with focus on the correlation between AST-evoked regulation of GRP and calpain activation.
  
  
  METHODS
  The effects of AST on GRP and apoptotic activity were assessed in HCT 116 human colon adenocarcinoma cells. Calpain activity was examined by using a fluorescence assay kit. Immunofluorescence staining and immunoprecipitation were employed to determine the localization and association between calpains and GRP. GRP78 gene silencing was performed to confirm the importance of GRP in anticancer drug activities. The modulation of GRP and calpains was also studied in nude mice xenograft.
  
  
  RESULTS
  ER stress-mediated apoptosis was induced by AST, as shown by elevation in both spliced XBP-1 and CHOP levels, with parallel up-regulation of GRP. The expression of XBP-1 and CHOP continued to increase after the peak level of GRP was attained at 24 h. Nevertheless, the initial increase in calpain activity as well as calpain I and II protein level was gradually declined at later stage of drug treatment. Besides, the induction of GRP was partly reversed by calpain inhibitors, with concurrent promotion of AST-mediated apoptosis. The knockdown of GRP78 by gene silencing resulted in higher sensitivity of colon cancer cells to AST-induced apoptosis and reduction of colony formation. The association between calpains and GRP78 had been confirmed by immunofluorescence staining and immunoprecipitation. Modulation of GRP and calpains by AST was similarly demonstrated in nude mice xenograft, leading to significant inhibition of tumor growth.
  
  
  CONCLUSIONS
  Our findings exemplify that calpains, in particular calpain II, play a permissive role in the modulation of GRP78 and consequent regulation of ER stress-induced apoptosis. Combination of calpain inhibitors and AST could exhibit a more pronounced pro-apoptotic effect. These results help to envisage a new therapeutic approach in colon cancer by targeting calpain and GRP.
authors.forename: Yue
authors.initials: Y
authors.lastname: Wang
authors.forename: Kathy K
authors.initials: KK
authors.lastname: Auyeung
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Hong Kong, China. jksko@hkbu.edu.hk.
authors.forename: Joshua K
authors.initials: JK
authors.lastname: Ko
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D027883:Astragalus Plant / Q000737:chemistry*; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D000091342:Endoplasmic Reticulum Chaperone BiP; D045325:HCT116 Cells; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D010936:Plant Extracts / Q000008:administration & dosage*; D012503:Saponins / Q000008:administration & dosage*; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; C000717248:Hspa5 protein, mouse; D008565:Membrane Proteins; D010936:Plant Extracts; D012503:Saponins; C018551:glucose-regulated proteins; D002154:Calpain