title: BH3-mimetic gossypol-induced autophagic cell death in mutant BRAF melanoma cells with high expression of p21Cip¹.).
abstract: OBJECTIVE
  The aim of the present study was to identify the potential therapeutic effects of BH3-mimetic gossypol on melanoma cells with acquired resistance to BRAF inhibitors.
  
  
  METHODS
  The IC50 values of gossypol were determined using MTT assays in three melanoma cell lines with different resistances to BRAF inhibitor. The effects of gossypol on three melanoma cell lines were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. The functional role of autophagy in gossypol-induced growth inhibition was investigated using siRNA-mediated knockdown of Beclin-1.
  
  
  RESULTS
  Gossypol retained its efficacy in BRAF-V600E melanoma clones with acquired resistance to BRAF inhibitors through a mechanism independent of MEK-ERK inhibition. Gossypol caused G2/M arrest in both BRAF mutant A375P and A375P/Mdr cells with high expression of p21(Cip1), regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21(Cip1) expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells. In particular, alleviation of autophagy by knockdown of Beclin-1 partially caused a resistance to gossypol-induced cell cycle arrest at G2/M in BRAF-V600E cells with a concomitant decreased induction of apoptosis.
  
  
  CONCLUSIONS
  Taken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21(Cip1) is a prerequisite for a positive response to its clinical application.
authors.affiliation: Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 406-772, Republic of Korea.
authors.forename: Gun-Hee
authors.initials: GH
authors.lastname: Jang
authors.affiliation: Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 406-772, Republic of Korea. Electronic address: mikelee@incheon.ac.kr.
authors.forename: Michael
authors.initials: M
authors.lastname: Lee
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D040761:Biomimetic Materials / Q000494:pharmacology; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006072:Gossypol / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D059566:M Phase Cell Cycle Checkpoints / Q000187:drug effects; D008545:Melanoma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics*; D034741:RNA, Small Interfering / Q000008:administration & dosage
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D008565:Membrane Proteins; D034741:RNA, Small Interfering; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D006072:Gossypol
keywords: Autophagy; BRAF inhibitor-resistance; Gossypol; Melanoma; p21(Cip1)