Journal
CANCER LETTERS
Volume 370, Issue 1, Pages 19-26Publisher
ELSEVIER IRELAND LTD
DOI: 10.1016/j.canlet.2015.10.008
Keywords
Gossypol; ATG5 knockout; Autophagy; CRISPR/Cas9
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Funding
- Basic Science Research Program through the National Research Foundation of Korea (NRF) - Ministry of Education, Science and Technology [2013R1A1A2A10058897]
- Incheon National University Research Grant
- National Research Foundation of Korea [2013R1A1A2A10058897] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Casmediated genome editing. The MU assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
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