Inactivation of ras and changes of mitochondrial membrane potential contribute to oridonin-induced autophagy in a431 cells.
We have previously shown that oridonin isolated from Rabdosia rubescens augmented apoptosis while inhibiting autophagy within 24 h in HeLa cells. However, the mechanisms between apoptosis and autophagy induced by oridonin in A431 cells are largely unknown. Here, it was found that autophagic level is significantly upregulated when A431 cells are pretreated with manumycin A (Ras specific inhibitor) compared with oridonin alone treatment, whereas cells precultured with GW5074 (Raf inhibitor) or PD98059 (ERK inhibitor) did not exhibit such an effect. Ras, but not Raf or ERK, was engaged in the control of oridonin-induced autophagy. At the same time, manumycin A contributes to oridonin-induced downregulation of Ras protein expression. Treatment with the combination of oridonin and manumycin A downregulated phosphorylation of Akt, downstream of phosphatidylinositol 3-OH kinase (PI3-K). Preincubation with the PI3-K inhibitor wortmannin and Akt inhibitor KP372-1 enhanced oridonin-induced apoptosis, whereas it inhibited oridonin-induced autophagy. However, under oridonin treatment, the expression of Beclin-1, which has autophagy-inducing activity, was reduced, suggesting that Beclin-1 did not participate in the oridonin-induced autophagy. Morphologic observations, DNA fragmentation analysis, and LDH activity-based assay showed that 3-methyladenine (3-MA), an inhibitor of autophagy, increased the apoptotic sensitivity of A431 cells to oridonin. In addition, manumycin A contributed to oridonin-induced decrease of mitochondrial membrane potential (Deltapsim), consistent with the upregulation of Bax/Bcl-2 ratio. In conclusion, Ras negatively regulated autophagy in oridonin-treated A431 cells, which might be associated with activation of class I PI3-K. Downregulation of Deltapsim and increasing of the ratio of Bax/Bcl-2 might also be partially responsible for the initiation of the autophagic process.
Li
Dan
D
China-Japan Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
Cui
Qiao
Q
Chen
Shen-geng
SG
Wu
Li-jun
LJ
Tashiro
Shin-ichi
S
Onodera
Satoshi
S
Ikejima
Takashi
T
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004224:Diterpenes / Q000737:chemistry / Q000494:pharmacology*; D045786:Diterpenes, Kaurane / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D015394:Molecular Structure; D010636:Phenols / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011090:Polyenes / Q000494:pharmacology; D053284:Polyunsaturated Alkamides / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors; D000077191:Wortmannin; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000225:Adenine; D000730:Androstadienes; D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; D004224:Diterpenes; D045786:Diterpenes, Kaurane; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D010636:Phenols; D010714:Phosphatidylethanolamines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011090:Polyenes; D053284:Polyunsaturated Alkamides; D019908:Proto-Oncogene Proteins c-raf; D000077191:Wortmannin; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D018631:ras Proteins; C494826:BAX protein, human; C491997:BECN1 protein, human; C477012:MAP1LC3A protein, human; C011959:oridonin; C483858:phosphatidylethanolamine; C025946:3-methyladenine; C054474:manumycin; C489251:5-iodo-3-((3,5-dibromo-4-hydroxyphenyl)methylene)-2-indolinone; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one