title: Autophagy, cell death and sustained senescence arrest in B16/F10 melanoma cells and HCT-116 colon carcinoma cells in response to the novel microtubule poison, JG-03-14.
abstract: OBJECTIVE
  Previous studies have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to kill breast tumor cells primarily through the promotion of autophagy. The current work was designed to determine whether autophagy was, in fact, the primary mode of action as well as susceptibility to JG-03-14 in two additional tumor cell models, the B16/F10 murine melanoma cell line and the HCT-116 human colon cancer cell line.
  
  
  METHODS
  Drug cytotoxicity was monitored based on viable cell number and clonogenic survival. Apoptosis was assessed by DAPI staining, the TUNEL assay and/or FACS analysis. Autophagy was monitored based on staining with acridine orange, redistribution and punctuation of RFP-LC3 and electron microscopy as well as p62 degradation. Senescence was evaluated based on β-galactosidase staining and alterations in cell morphology. Drug effects were also evaluated in a murine model of B16/F10 cells that localizes to the lungs while peripheral neuropathy was assessed by three complementary behavioral assays.
  
  
  RESULTS
  Both HCT-116 colon cancer cells and B16/F10 melanoma cells were sensitive to JG-03-14 in that the drug demonstrated tumor cell killing. However, there was minimal induction of apoptosis. In contrast, there was clear evidence for autophagy and autophagic flux while the residual surviving cells appeared to be in a state of irreversible senescence. Inhibition of drug-induced autophagy in either the melanoma cells or the colon carcinoma cells was only slightly protective as the cells instead died by apoptosis. JG-03-14 reduced the size of tumor nodules in mice lungs; furthermore, the drug did not promote peripheral neuropathy.
  
  
  CONCLUSIONS
  Taken together with evidence for its actions as a vascular disrupting agent, these observations support the potential utility of JG-03-14 to effectively treat malignancies that might be resistant to conventional chemotherapy through evasion of apoptosis.
authors.affiliation: Department of Pharmacology and Pharmacology, Massey Cancer Center, Virginia Commonwealth University, PO Box 980035, Richmond, VA 23298, USA.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Biggers
authors.forename: Tuyen
authors.initials: T
authors.lastname: Nguyen
authors.forename: Xu
authors.initials: X
authors.lastname: Di
authors.forename: John T
authors.initials: JT
authors.lastname: Gupton
authors.forename: Scott C
authors.initials: SC
authors.lastname: Henderson
authors.forename: Sean M
authors.initials: SM
authors.lastname: Emery
authors.forename: Moureq
authors.initials: M
authors.lastname: Alotaibi
authors.forename: Kimber L
authors.initials: KL
authors.lastname: White
authors.forename: Ronetta
authors.initials: R
authors.lastname: Brown
authors.forename: Jorge
authors.initials: J
authors.lastname: Almenara
authors.forename: David A
authors.initials: DA
authors.lastname: Gewirtz
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D016922:Cellular Senescence / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D045325:HCT116 Cells; D006801:Humans; D008546:Melanoma, Experimental / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008870:Microtubules / Q000187:drug effects*; D010523:Peripheral Nervous System Diseases / Q000139:chemically induced; D011758:Pyrroles / Q000494:pharmacology* / Q000633:toxicity
chemical_list: C521391:3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester; D011758:Pyrroles