title: Effects of PHA-665752 and Cetuximab Combination Treatment on In Vitro and Murine Xenograft Growth of Human Colorectal Cancer Cells with KRAS or BRAF Mutations.
abstract: It remains unknown whether blockade of c-Met signaling and epidermal growth factor receptor signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. In this study, we investigated the effects of the c-Met inhibitor PHA-665752 alone and in combination with cetuximab on the growth of human CRC cells in vitro and in mouse xenografts.
  
  
  
  Human CRC cell lines (Caco2, HCT-116, and HT-29) and mice bearing HCT-116 xenografts were treated with cetuximab in the absence or presence of PHA-665752. Cell viability and apoptosis were examined using the MTT and TUNEL assays, respectively. Vimentin was measured by immunohistochemistry as a marker for epithelial-to-mesenchymal transition. Western blotting was used to determine signaling protein expression levels.
  
  
  
  The MTT assay showed that the growth of Caco2, HCT-116, and HT-29 cells was inhibited by PHA-665752 in a dose-dependent manner, but only Caco2 cell growth was suppressed by cetuximab. Combination treatment with PHA-665752 and cetuximab inhibited the proliferation of Caco2 cells and RAS mutant CRC cell lines. However, relative to the PHA-665752-alone treatment group, HT-29 cells with a BRAF mutation showed no noticeable effect. The mean tumor volume in mice treated with cetuximab in combination with PHA-665752 was significantly smaller than that in the mice treated with only cetuximab (P = 0.033) or PHA-665752 (P < 0.01). Similarly, the expression of vimentin in the mice treated with PHA-665752 in combination with cetuximab was significantly lower than that in the mice treated with cetuximab or PHA-665752 alone (P < 0.05 in each case). TUNEL assays revealed that treatment with PHA-665752 in combination with cetuximab markedly increased CRC cell apoptosis. Western blotting analysis of signaling protein expression showed that PHA- 665752 inhibited Met phosphorylation (P < 0.05). In addition, treatment with cetuximab alone or in combination with PHA-665752 effectively inhibited EGFR phosphorylation (P < 0.05).
  
  
  
  Combination treatment with PHA-665752 and cetuximab suppressed in vitro and in vivo CRC cell growth more than treatment with either agent alone did.
authors.affiliation: Department of Oncology, Hebei General Hospital, Shijiazhuang 050051, Hebei, China.
authors.forename: Yi-Tao
authors.initials: YT
authors.lastname: Jia
authors.affiliation: Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.|Department of Emergency Medicine, the Second Affiliated Hospital of Xingtai Medical College, Xingtai 054000, Hebei, China.
authors.forename: Dong-Hai
authors.initials: DH
authors.lastname: Yang
authors.affiliation: Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.
authors.forename: Zhaolong
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Bi
authors.affiliation: Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.
authors.forename: Wei-Hua
authors.initials: WH
authors.lastname: Han
authors.affiliation: Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Feng
authors.affiliation: Department of Oncology, Hebei General Hospital, Shijiazhuang 050051, Hebei, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhi
authors.affiliation: Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Gu
authors.affiliation: Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Duan
authors.affiliation: Experimental Animal Center, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, Hebei, China.
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Wu
authors.affiliation: Experimental Animal Center, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, Hebei, China.
authors.forename: Ying-Chao
authors.initials: YC
authors.lastname: Ju
authors.affiliation: Department of Pharmacy, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, Hebei, China.
authors.forename: Ming-Xia
authors.initials: MX
authors.lastname: Wang
authors.affiliation: Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.
authors.forename: Zhong-Xin
authors.initials: ZX
authors.lastname: Li
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis; D049109:Cell Proliferation; D000068818:Cetuximab / Q000008:administration & dosage; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000008:administration & dosage; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D013450:Sulfones / Q000008:administration & dosage; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: C480541:5-((2,6-dichlorobenzyl)sulfonyl)-3-((3,5-dimethyl-4-((2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)carbonyl)-1H-pyrrol-2-yl)methylene)-1,3-dihydro-2H-indol-2-one; D007211:Indoles; C117307:KRAS protein, human; D013450:Sulfones; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D000068818:Cetuximab
keywords: BRAF; CRC cell lines; Colorectal cancer; RAS; c-Met; cetuximab.