Thioredoxin-1 phosphorylated at T100 is needed for its anti-apoptotic activity in HepG2 cancer cells.
OBJECTIVE
Thioredoxin-1 (Trx) is an important protein involved in the regulation of apoptosis and in enhancing drug resistance in cancer cells. Threonine100 (T100) of Trx was reported to be phosphorylated; however, the role of this phosphorylation in regulating activity remains unresolved. We explored whether and how the phosphorylation of Trx is involved in drug resistance in cancer cells.


METHODS
The levels of phosphorylated Trx were detected by sandwich ELISA. Cell viability was investigated using Alamar Blue assay, and apoptosis was evaluated with Hoechst33258 staining. Western blotting was used to examine the changes in the expression levels of Trx, NF-kappaB p65 subunit, ASK-1 and 6His tag. Additionally, we took advantage of phorbol-12-myristate-13-acetate (PMA)to activate protein kinase C (PKC) and staurosporine to inhibit PKC.


RESULTS
Trx mutated at T100 causes lower survival rate induced by H(2)O(2), and higher apoptosis rate induced by cis-platinum and adriamycin in HepG2 cells. T100 of Trx can be phosphorylated through the PKC-dependent pathway. Furthermore, the resistance of anticancer drugs can be decreased when the phosphorylation of T100 in Trx was blocked by staurosporine. Though the Trx-ASK1 complex is not affected, the phosphorylation contributes to the nuclear location of Trx, and then up-regulates the activity of NF-kappaB.


CONCLUSIONS
It was firstly found that the phosphorylation of Trx at T100 plays an important role in its cytoprotective activity in cancer cells. Thus, besides blocking the active site of Trx, inhibiting the phosphorylation of Trx at T100 may be a new potential cancer therapy target.
Chen
Xiaoping
X
Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Tang
Wenxin
W
Liu
Shan
S
Yu
Longjiang
L
Chen
Zhengwang
Z
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D056945:Hep G2 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D013912:Threonine; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human