Thioredoxin-1 phosphorylated at T100 is needed for its anti-apoptotic activity in HepG2 cancer cells

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.

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.

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.

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.