Inhibition of lysosome degradation on autophagosome formation and responses to GMI, an immunomodulatory protein from Ganoderma microsporum.
OBJECTIVE
Autophagic cell death is considered a self-destructive process that results from large amounts of autophagic flux. In our previous study, GMI, a recombinant fungal immunomodulatory protein cloned from Ganoderma microsporum, induced autophagic cell death in lung cancer cells. The aim of this study was to examine the role of autophagosome accumulation in GMI-mediated cell death.


METHODS
Western blot analysis, flow cytometry and confocal microscopy were used to evaluate the effects of different treatments, including silencing of ATP6V0A1 by use of short hairpin RNAi, on GMI-mediated cell death, lung cancer cell viability and autophagosome accumulation in vitro.


RESULTS
Lysosome inhibitors bafilomycin-A1 and chloroquine increased GMI-mediated autophagic cell death. GMI and bafilomycin-A1 co-treatment induced the accumulation of large amounts of autophagosomes, but did not significantly induce apoptosis. GMI elicited autophagy through the PKB (Akt)/mammalian target of rapamycin signalling pathway. Silencing of ATP6V0A1, one subunit of vesicular H(+)-ATPases (V-ATPases) that mediates lysosome acidification, spontaneously induced autophagosome accumulation, but did not affect lysosome acidity. GMI-mediated autophagosome accumulation and cytotoxicity was increased in shATP6V0A1 lung cancer cells. Furthermore, ATP6V0A1 silencing decreased autophagosome and lysosome fusion in GMI-treated CaLu-1/GFP-LC3 lung cancer cells.


CONCLUSIONS
We demonstrated that autophagosome accumulation induces autophagic cell death in a GMI treatment model, and ATP6V0A1 plays an important role in mediating autophagosome-lysosome fusion. Our findings provide new insights into the mechanisms involved in the induction of autophagic cell death.
Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan.
I-Lun
IL
Hsin
Gwo-Tarng
GT
Sheu
Ming-Shiou
MS
Jan
Hai-Lun
HL
Sun
Tzu-Chin
TC
Wu
Ling-Yen
LY
Chiu
Ko-Huang
KH
Lue
Jiunn-Liang
JL
Ko
D001343:Autophagy / Q000187:drug effects; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology; D005656:Fungal Proteins / Q000494:pharmacology*; D032902:Ganoderma; D020868:Gene Silencing; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology*; D008247:Lysosomes / Q000187:drug effects; D018942:Macrolides / Q000494:pharmacology; D010588:Phagosomes / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics
D005656:Fungal Proteins; D007155:Immunologic Factors; D018942:Macrolides; D002738:Chloroquine; C040929:bafilomycin A1; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; C529316:ATP6V0A1 protein, human; D025262:Vacuolar Proton-Translocating ATPases