| The thioredoxins are ubiquitous proteins containing conserved Cys-Gly-Pro-Cys redox catalytic site. Mammalian thioredoxin family members include thioredoxin-1 (Trx-1), and mitochondrial thioredoxin-2 (Trx-2). Thioredoxin is reduced by NADPH and thioredoxin reductase and, in turn reduces oxidized cysteine groups on proteins. When thioredoxin levels are elevated there is increased cell growth and resistance to the normal mechanism of programmed cell death. An increase in thioredoxin levels seen in many human primary cancers compared to normal tissue appears to contribute to increased cancer cell growth and resistance to chemotherapy. Mechanisms by which thioredoxin increases cell growth include an increased supply of reducing equivalents for DNA synthesis, activation of transcription factors that regulate cell growth, and an increase in the sensitivity of cells to other cytokines and growth factors. The mechanisms for the inhibition of apoptosis by thioredoxin are just now being elucidated. Because of its role in stimulating cancer cell growth and as an inhibitor of apoptosis, thioredoxin offers a target for the development of drugs to treat and prevent cancer.Our studies are as following:1) Trx-1 gene was obtained by RT-PCR. Prokaryotic fusion gene expression vector, pET32a-Trx, was constructed. A high level of expression of fusion protein in E.coli was detected after IPTG induction and purified proteins were obtained by affinity chromatography.2) The purified Trx-1 proteins were mixed with Freund's complete or incomplete adjuvant as antigen to immune rabbits. ELISA assay revealed that the titer of the prepared antiserum against Trx-1 protein was as high as 1:10000. IgG was purified with saturated ammonium sulfate. These polyclonal antibodies are special to Trx-1 and can be used to detect Trx-1. 3) The contrations of Trx-1 in the serum of breast cancer patients were detected with ELISA, and we found that the contration of Trx-1 is a little lower than control.4) Trx-1 gene was cloned into eukaryotic expression vector, pcDNA3.1+ and transferred into breast cancer cells, MCF-7.5) Trx-1 overexpressed in MCF-7 cells after dealed with Vc or transferred with Trx-1 cDNA. At the same time, more cells were in S phase. Meanwhile, higher Trx-1 in vitro can also bring more cells into S phage. So Trx-1 contributes to prolonging S phase of MCF-7 cells, both in vivo and in vitro.6) We explored the association of the nuclear Trx-1 with the cisplatin-mediated apoptosis of breast cancer cells MCF-7. Firstly, we found that higher total Trx-1 accompanied by no change of nuclear Trx-1 can not influence apoptosis induced by cisplatin in MCF-7 cells transferred with Trx-1 cDNA. Secondly, higher nuclear Trx-1 accompanied by no change of total Trx-1 can protect cells from apoptosis induced by cisplatin. Thirdly, high nuclear Trx-1 involves in the cisplatin-resistance in cisplatin-resistive cells. Meanwhile, we found that the mRNA level of p53 is closely correlated with the level of nuclear Trx-1. In summary, we concluded that the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53.Based our data, we can include that i) the contrations of Trx-1 in the serum of breast cancer patients is a little lower than control, ii) Trx-1 contributes to prolonging S phase of MCF-7 cells, both in vivo and in vitro, and iii) the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53.
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